GcatWiki - User contributions [en]

Golden Gate Assembly Protocol for BsmB1

2018-06-14T12:38:29Z

Kyroland:

Golden Gate Assembly Protocol for BsmB1
GGA mixture contains:

:1 µL (50 ng) Plasmid

:1 µL promoter or other insert

:1 µL 10X Promega Ligase Buffer

:6 µL dH2O

:0.5 µL BsmB1 high fidelity (HF) restriction enzyme

:9.5 µL volume

Place tubes into 55°C water bath for 15 minutes. Add 0.5 µL T4 DNA Ligase to the mixture.

Turn on the PCR machine. Put tube into machine. Program it for the following cycles:

:• 20 cycles

:• 55°C for 10 minutes

:• 37°C for 1 minute

:• 16°C for 1 minute

:• 22°C holding temperature

This DNA ligation is ready for transformation. You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.

Transformation of GGA BsmB1
For each reaction done you will need an agar plate and 50µL of competent cells mixed with competent cell buffer. Mix the 50µL of cell mixture with the GGA product. Place the entire content of the tube on the plate, spread and incubate.

Ky Roland

2018-03-27T14:39:16Z

Kyroland: /* Transcription Factors */

*25 and 24 = paired reads, Male mouse 2078, C201R 
'''23 and 22 = paired reads, Female mouse 2077, WT''' 
21 and 20 = paired reads, Male mouse 2076, WT 
19 and 18 = paired reads, Male mouse 2075, WT 
17 and 16 = paired reads, Male mouse 2074, WT 
*15 and 14 = paired reads, Male mouse 2073, C201R 
*'''13 and 12 = paired reads, Female mouse 2072, C201R''' 
*'''11 and 10 = paired reads, Female mouse 2071, C201R''' 
*'''9 and 8 = paired reads, Female mouse 2070, C201R''' 
'''7 and 6 = paired reads, Female mouse 2081, WT''' 
'''5 and 4 = paired reads, Female mouse 2080, WT''' 
*3 and 2 = paired reads, Male mouse 2079, C201R 

'''DESeq2- Comparison of WT vs. Mutant Females: Named-Types''' 
*Mutant will be in numerator, and WT will be int he denominator; a number > 1 will mean that there is an overexertion in the wild type.
*We should not expect differential expression because the papers showed no significant differential mRNA expression between the mutant and wild type.

'''Significant Genes:''' 
*'''Sphk2'''- [http://www.genecards.org/cgi-bin/carddisp.pl?gene=Sphk2 Gene Cards ] 
** Bears a relationship to Polr2a, another gene included in our list, but not significant 

Ontological Discovery Environment ID- What GeneWeaver uses 
*A common entity that gene species can map to in geneweaver 
*to develop sets that you can match 

Types of functional Genomics Data:

Mapping Data 
* QTL Positional Candidates 
*GWAS Candidate 

Expression Data: From NIF 
*Drug Related Genes Database- from literature 
*Allen Brain Atlas- Mouse 
** ensure localized expression 

View similar gene sets 
Hierarchical similarity graph- 
*Top nodes are the most highly connected nodes, with the most highly connected gene being red 

'''How to use GeneWeaver:''' 
[http://www.beta.geneweaver.org GeneWeaver Beta] 
* Search a key term and data related to your search term will appear ranked by relevance 
*You can use boolean queries to augment your search results to be more or less inclusive 

HiSim- a sort of multiway venn diagram without overlapping results of high level intersections of gene sets 
*Shows various combinations of intersecting gene sets 
*Emphasizes a relationship between the sets 

Gene Set Graph Tool 
*Different way of looking at data comparisons made in HiSim, but more useful for emphasizing the genes 

Jacard Similarity- 
View pairwise similarities and overlap 

no method for two different score types- can upload gene sets twice using different score types and then threshold them in analysis 

Might have to convert Gene identifiers if they are mixed in the gene set 

'''Gene Weaver Tips:''' 
*Use Gene ID's as the gene identifier 
----

== '''Transcription Factors''' ==
Start with Gene search in [https://www.ncbi.nlm.nih.gov/gene/2516 NCBI]. 

Where is this expressed? (all 4 data sets) 

Human conditions when mutated? 

How many proteins does NR5A1 interact with? 

What GO terms are associated with NR5A1? 

'''nhr-25: Model Transcription Factor''' 
*Background 
**an important developmental regulator 
**known phenotypes and tools 
**mammalian orthologs 

*NHR-25 
**transcription factor of large family: nuclear hormone receptors 
**expressed in seam and vulval cells throughout development 
**'''human orthologs''': SF-1 and LRH-1; and '''Drosophila''': Ftz-F1 

*Mutants to identify regulated genes 
**nhr-25 is essential, therefore we have to knockdown the gene as opposed to knocking it out 
**transiently knocked down by RNAi- molting and vulval defects 

**nhr-25(ku217) mutant- molting and vulval defect 
***leucine to proline point mutation in the D 
NA biding domain 
***mutant has reduced DNA binding 

*identification of nhr-25 regulated genes 
**sequencing library consists of 10 worms from roughly the same developmental stage 

'''Pairwise Comparisons:'''
*WT vs Mutant [http://cbl-gorilla.cs.technion.ac.il/GOrilla/esword6j/GOResults.html GOrilla] 
*WT vs. Knockdown [http://cbl-gorilla.cs.technion.ac.il/GOrilla/1asdvat3/GOResults.html GOrilla] 
*Mutant vs. Knockdown 

'''Thought Process''' 
*Narrow down list using adjusted p-value (cutoff of .01) 
*Rank gene list based on fold change (largest to smallest) 
*Input 3 ranked gene list into gene Venn 
*Analyze list of genes from Gene Venn that show common genes from differential expression from all three DE comparisons 
*Alanyze gene list of genes from Gene Venn that showgenes only expressed in WT vs. Mutant and not other 2 DE comparisons 
**Highest enrich
*Knockdown- point mutation, mRNA is still in tact 
*RNAi- binds to mRNA and mRNA is destroyed; no protein is created 
*Compare genes expressed in up regulated vs. down regulated genes and do GOrilla and WormBase analysis

*Vulva and cuticle are two phenotypes that we know are affected (known phenotypes) in the mutant and knockdown worms 
*Wormbase limitation, it is comparing larger categories 
Venn Diagram tool: [http://genevenn.sourceforge.net Gene venn]

Ky Roland

2018-03-27T13:51:20Z

Kyroland: /* Transcription Factors */

*25 and 24 = paired reads, Male mouse 2078, C201R 
'''23 and 22 = paired reads, Female mouse 2077, WT''' 
21 and 20 = paired reads, Male mouse 2076, WT 
19 and 18 = paired reads, Male mouse 2075, WT 
17 and 16 = paired reads, Male mouse 2074, WT 
*15 and 14 = paired reads, Male mouse 2073, C201R 
*'''13 and 12 = paired reads, Female mouse 2072, C201R''' 
*'''11 and 10 = paired reads, Female mouse 2071, C201R''' 
*'''9 and 8 = paired reads, Female mouse 2070, C201R''' 
'''7 and 6 = paired reads, Female mouse 2081, WT''' 
'''5 and 4 = paired reads, Female mouse 2080, WT''' 
*3 and 2 = paired reads, Male mouse 2079, C201R 

'''DESeq2- Comparison of WT vs. Mutant Females: Named-Types''' 
*Mutant will be in numerator, and WT will be int he denominator; a number > 1 will mean that there is an overexertion in the wild type.
*We should not expect differential expression because the papers showed no significant differential mRNA expression between the mutant and wild type.

'''Significant Genes:''' 
*'''Sphk2'''- [http://www.genecards.org/cgi-bin/carddisp.pl?gene=Sphk2 Gene Cards ] 
** Bears a relationship to Polr2a, another gene included in our list, but not significant 

Ontological Discovery Environment ID- What GeneWeaver uses 
*A common entity that gene species can map to in geneweaver 
*to develop sets that you can match 

Types of functional Genomics Data:

Mapping Data 
* QTL Positional Candidates 
*GWAS Candidate 

Expression Data: From NIF 
*Drug Related Genes Database- from literature 
*Allen Brain Atlas- Mouse 
** ensure localized expression 

View similar gene sets 
Hierarchical similarity graph- 
*Top nodes are the most highly connected nodes, with the most highly connected gene being red 

'''How to use GeneWeaver:''' 
[http://www.beta.geneweaver.org GeneWeaver Beta] 
* Search a key term and data related to your search term will appear ranked by relevance 
*You can use boolean queries to augment your search results to be more or less inclusive 

HiSim- a sort of multiway venn diagram without overlapping results of high level intersections of gene sets 
*Shows various combinations of intersecting gene sets 
*Emphasizes a relationship between the sets 

Gene Set Graph Tool 
*Different way of looking at data comparisons made in HiSim, but more useful for emphasizing the genes 

Jacard Similarity- 
View pairwise similarities and overlap 

no method for two different score types- can upload gene sets twice using different score types and then threshold them in analysis 

Might have to convert Gene identifiers if they are mixed in the gene set 

'''Gene Weaver Tips:''' 
*Use Gene ID's as the gene identifier 
----

== '''Transcription Factors''' ==
Start with Gene search in [https://www.ncbi.nlm.nih.gov/gene/2516 NCBI]. 

Where is this expressed? (all 4 data sets) 

Human conditions when mutated? 

How many proteins does NR5A1 interact with? 

What GO terms are associated with NR5A1? 

'''nhr-25: Model Transcription Factor''' 
*Background 
**an important developmental regulator 
**known phenotypes and tools 
**mammalian orthologs 

*NHR-25 
**transcription factor of large family: nuclear hormone receptors 
**expressed in seam and vulval cells throughout development 
**'''human orthologs''': SF-1 and LRH-1; and '''Drosophila''': Ftz-F1 

*Mutants to identify regulated genes 
**nhr-25 is essential, therefore we have to knockdown the gene as opposed to knocking it out 
**transiently knocked down by RNAi- molting and vulval defects 

**nhr-25(ku217) mutant- molting and vulval defect 
***leucine to proline point mutation in the D 
NA biding domain 
***mutant has reduced DNA binding 

*identification of nhr-25 regulated genes 
**sequencing library consists of 10 worms from roughly the same developmental stage 

'''Pairwise Comparisons:'''
*WT vs Mutant [http://cbl-gorilla.cs.technion.ac.il/GOrilla/esword6j/GOResults.html GOrilla] 
*WT vs. Knockdown [http://cbl-gorilla.cs.technion.ac.il/GOrilla/1asdvat3/GOResults.html GOrilla] 
*Mutant vs. Knockdown 

'''Thought Process''' 
*Narrow down list using adjusted p-value (cutoff of .01) 
*Rank gene list based on fold change (largest to smallest) 
*Knockdown- moint mutation, mRNA is still in tact 
*RNAi- binds to mRNA and mRNA is destroyed; no protein is created 
*Compare genes expressed in up regulated vs. down regulated genes and do GOrilla and WormBase analysis

*Vulva and cuticle are two phenotypes that we know are affected (known phenotypes) in the mutant and knockdown worms 
*Wormbase limitation, it is comparing larger categories 
Venn Diagram tool: [http://genevenn.sourceforge.net Gene venn]

Ky Roland

2018-03-20T14:58:18Z

Kyroland:

*25 and 24 = paired reads, Male mouse 2078, C201R 
'''23 and 22 = paired reads, Female mouse 2077, WT''' 
21 and 20 = paired reads, Male mouse 2076, WT 
19 and 18 = paired reads, Male mouse 2075, WT 
17 and 16 = paired reads, Male mouse 2074, WT 
*15 and 14 = paired reads, Male mouse 2073, C201R 
*'''13 and 12 = paired reads, Female mouse 2072, C201R''' 
*'''11 and 10 = paired reads, Female mouse 2071, C201R''' 
*'''9 and 8 = paired reads, Female mouse 2070, C201R''' 
'''7 and 6 = paired reads, Female mouse 2081, WT''' 
'''5 and 4 = paired reads, Female mouse 2080, WT''' 
*3 and 2 = paired reads, Male mouse 2079, C201R 

'''DESeq2- Comparison of WT vs. Mutant Females: Named-Types''' 
*Mutant will be in numerator, and WT will be int he denominator; a number > 1 will mean that there is an overexertion in the wild type.
*We should not expect differential expression because the papers showed no significant differential mRNA expression between the mutant and wild type.

'''Significant Genes:''' 
*'''Sphk2'''- [http://www.genecards.org/cgi-bin/carddisp.pl?gene=Sphk2 Gene Cards ] 
** Bears a relationship to Polr2a, another gene included in our list, but not significant 

Ontological Discovery Environment ID- What GeneWeaver uses 
*A common entity that gene species can map to in geneweaver 
*to develop sets that you can match 

Types of functional Genomics Data:

Mapping Data 
* QTL Positional Candidates 
*GWAS Candidate 

Expression Data: From NIF 
*Drug Related Genes Database- from literature 
*Allen Brain Atlas- Mouse 
** ensure localized expression 

View similar gene sets 
Hierarchical similarity graph- 
*Top nodes are the most highly connected nodes, with the most highly connected gene being red 

'''How to use GeneWeaver:''' 
[http://www.beta.geneweaver.org GeneWeaver Beta] 
* Search a key term and data related to your search term will appear ranked by relevance 
*You can use boolean queries to augment your search results to be more or less inclusive 

HiSim- a sort of multiway venn diagram without overlapping results of high level intersections of gene sets 
*Shows various combinations of intersecting gene sets 
*Emphasizes a relationship between the sets 

Gene Set Graph Tool 
*Different way of looking at data comparisons made in HiSim, but more useful for emphasizing the genes 

Jacard Similarity- 
View pairwise similarities and overlap 

no method for two different score types- can upload gene sets twice using different score types and then threshold them in analysis 

Might have to convert Gene identifiers if they are mixed in the gene set 

'''Gene Weaver Tips:''' 
*Use Gene ID's as the gene identifier 
----

== '''Transcription Factors''' ==
Start with Gene search in [https://www.ncbi.nlm.nih.gov/gene/2516 NCBI]. 

Where is this expressed? (all 4 data sets) 

Human conditions when mutated? 

How many proteins does NR5A1 interact with? 

What GO terms are associated with NR5A1? 

'''nhr-25: Model Transcription Factor''' 
*Background 
**an important developmental regulator 
**known phenotypes and tools 
**mammalian orthologs 

*NHR-25 
**transcription factor of large family: nuclear hormone receptors 
**expressed in seam and vulval cells throughout development 
**'''human orthologs''': SF-1 and LRH-1; and '''Drosophila''': Ftz-F1 

*Mutants to identify regulated genes 
**nhr-25 is essential, therefore we have to knockdown the gene as opposed to knocking it out 
**transiently knocked down by RNAi- molting and vulval defects 

**nhr-25(ku217) mutant- molting and vulval defect 
***leucine to proline point mutation in the D 
NA biding domain 
***mutant has reduced DNA binding 

*identification of nhr-25 regulated genes 
**sequencing library consists of 10 worms from roughly the same developmental stage 

'''Pairwise Comparisons:'''
*WT vs Mutant [http://cbl-gorilla.cs.technion.ac.il/GOrilla/esword6j/GOResults.html GOrilla] 
*WT vs. Knockdown [http://cbl-gorilla.cs.technion.ac.il/GOrilla/1asdvat3/GOResults.html GOrilla] 
*Mutant vs. Knockdown 

'''Thought Process''' 
*Narrow down list using adjusted p-value (cutoff of .01) 
*Rank gene list based on fold change (largest to smallest) 
*Knockdown- moint mutation, mRNA is still in tact 
*RNAi- binds to mRNA and mRNA is destroyed; no protein is created 
*Compare genes expressed in up regulated vs. down regulated genes and do GOrilla analysis

Ky Roland

2018-03-20T14:16:23Z

Kyroland:

*25 and 24 = paired reads, Male mouse 2078, C201R 
'''23 and 22 = paired reads, Female mouse 2077, WT''' 
21 and 20 = paired reads, Male mouse 2076, WT 
19 and 18 = paired reads, Male mouse 2075, WT 
17 and 16 = paired reads, Male mouse 2074, WT 
*15 and 14 = paired reads, Male mouse 2073, C201R 
*'''13 and 12 = paired reads, Female mouse 2072, C201R''' 
*'''11 and 10 = paired reads, Female mouse 2071, C201R''' 
*'''9 and 8 = paired reads, Female mouse 2070, C201R''' 
'''7 and 6 = paired reads, Female mouse 2081, WT''' 
'''5 and 4 = paired reads, Female mouse 2080, WT''' 
*3 and 2 = paired reads, Male mouse 2079, C201R 

'''DESeq2- Comparison of WT vs. Mutant Females: Named-Types''' 
*Mutant will be in numerator, and WT will be int he denominator; a number > 1 will mean that there is an overexertion in the wild type.
*We should not expect differential expression because the papers showed no significant differential mRNA expression between the mutant and wild type.

'''Significant Genes:''' 
*'''Sphk2'''- [http://www.genecards.org/cgi-bin/carddisp.pl?gene=Sphk2 Gene Cards ] 
** Bears a relationship to Polr2a, another gene included in our list, but not significant 

Ontological Discovery Environment ID- What GeneWeaver uses 
*A common entity that gene species can map to in geneweaver 
*to develop sets that you can match 

Types of functional Genomics Data:

Mapping Data 
* QTL Positional Candidates 
*GWAS Candidate 

Expression Data: From NIF 
*Drug Related Genes Database- from literature 
*Allen Brain Atlas- Mouse 
** ensure localized expression 

View similar gene sets 
Hierarchical similarity graph- 
*Top nodes are the most highly connected nodes, with the most highly connected gene being red 

'''How to use GeneWeaver:''' 
[http://www.beta.geneweaver.org GeneWeaver Beta] 
* Search a key term and data related to your search term will appear ranked by relevance 
*You can use boolean queries to augment your search results to be more or less inclusive 

HiSim- a sort of multiway venn diagram without overlapping results of high level intersections of gene sets 
*Shows various combinations of intersecting gene sets 
*Emphasizes a relationship between the sets 

Gene Set Graph Tool 
*Different way of looking at data comparisons made in HiSim, but more useful for emphasizing the genes 

Jacard Similarity- 
View pairwise similarities and overlap 

no method for two different score types- can upload gene sets twice using different score types and then threshold them in analysis 

Might have to convert Gene identifiers if they are mixed in the gene set 

'''Gene Weaver Tips:''' 
*Use Gene ID's as the gene identifier 
----

== '''Transcription Factors''' ==
Start with Gene search in [https://www.ncbi.nlm.nih.gov/gene/2516 NCBI]. 

Where is this expressed? (all 4 data sets) 

Human conditions when mutated? 

How many proteins does NR5A1 interact with? 

What GO terms are associated with NR5A1? 

'''nhr-25: Model Transcription Factor''' 
*Background 
**an important developmental regulator 
**known phenotypes and tools 
**mammalian orthologs 

*NHR-25 
**transcription factor of large family: nuclear hormone receptors 
**expressed in seam and vulval cells throughout development 
**'''human orthologs''': SF-1 and LRH-1; and '''Drosophila''': Ftz-F1 

*Mutants to identify regulated genes 
**nhr-25 is essential, therefore we have to knockdown the gene as opposed to knocking it out 
**transiently knocked down by RNAi- molting and vulval defects 

**nhr-25(ku217) mutant- molting and vulval defect 
***leucine to proline point mutation in the D 
NA biding domain 
***mutant has reduced DNA binding 

*identification of nhr-25 regulated genes 
**sequencing library consists of 10 worms from roughly the same developmental stage 

'''Pairwise COmparisons:'''
*WT vs Mutant 
*WT vs. Knockdown 
*Mutant vs. Knockdown 

*Narrow down list using adjusted p-value (cutoff of .01) 
*Rank gene list based on fold change (largest to smallest) 
*Knockdown- moint mutation, mRNA is still in tact 
*RNAi- binds to mRNA and mRNA is destroyed; no protein is created

Ky Roland

2018-03-20T13:48:19Z

Kyroland:

*25 and 24 = paired reads, Male mouse 2078, C201R 
'''23 and 22 = paired reads, Female mouse 2077, WT''' 
21 and 20 = paired reads, Male mouse 2076, WT 
19 and 18 = paired reads, Male mouse 2075, WT 
17 and 16 = paired reads, Male mouse 2074, WT 
*15 and 14 = paired reads, Male mouse 2073, C201R 
*'''13 and 12 = paired reads, Female mouse 2072, C201R''' 
*'''11 and 10 = paired reads, Female mouse 2071, C201R''' 
*'''9 and 8 = paired reads, Female mouse 2070, C201R''' 
'''7 and 6 = paired reads, Female mouse 2081, WT''' 
'''5 and 4 = paired reads, Female mouse 2080, WT''' 
*3 and 2 = paired reads, Male mouse 2079, C201R 

'''DESeq2- Comparison of WT vs. Mutant Females: Named-Types''' 
*Mutant will be in numerator, and WT will be int he denominator; a number > 1 will mean that there is an overexertion in the wild type.
*We should not expect differential expression because the papers showed no significant differential mRNA expression between the mutant and wild type.

'''Significant Genes:''' 
*'''Sphk2'''- [http://www.genecards.org/cgi-bin/carddisp.pl?gene=Sphk2 Gene Cards ] 
** Bears a relationship to Polr2a, another gene included in our list, but not significant 

Ontological Discovery Environment ID- What GeneWeaver uses 
*A common entity that gene species can map to in geneweaver 
*to develop sets that you can match 

Types of functional Genomics Data:

Mapping Data 
* QTL Positional Candidates 
*GWAS Candidate 

Expression Data: From NIF 
*Drug Related Genes Database- from literature 
*Allen Brain Atlas- Mouse 
** ensure localized expression 

View similar gene sets 
Hierarchical similarity graph- 
*Top nodes are the most highly connected nodes, with the most highly connected gene being red 

'''How to use GeneWeaver:''' 
[http://www.beta.geneweaver.org GeneWeaver Beta] 
* Search a key term and data related to your search term will appear ranked by relevance 
*You can use boolean queries to augment your search results to be more or less inclusive 

HiSim- a sort of multiway venn diagram without overlapping results of high level intersections of gene sets 
*Shows various combinations of intersecting gene sets 
*Emphasizes a relationship between the sets 

Gene Set Graph Tool 
*Different way of looking at data comparisons made in HiSim, but more useful for emphasizing the genes 

Jacard Similarity- 
View pairwise similarities and overlap 

no method for two different score types- can upload gene sets twice using different score types and then threshold them in analysis 

Might have to convert Gene identifiers if they are mixed in the gene set 

'''Gene Weaver Tips:''' 
*Use Gene ID's as the gene identifier 
----

== '''Transcription Factors''' ==
Start with Gene search in [https://www.ncbi.nlm.nih.gov/gene/2516 NCBI]. 

Where is this expressed? (all 4 data sets) 

Human conditions when mutated? 

How many proteins does NR5A1 interact with? 

What GO terms are associated with NR5A1? 

'''nhr-25: Model Transcription Factor''' 
*Background 
**an important developmental regulator 
**known phenotypes and tools 
**mammalian orthologs 

*NHR-25 
**transcription factor of large family: nuclear hormone receptors 
**expressed in seam and vulval cells throughout development 
**'''human orthologs''': SF-1 and LRH-1; and '''Drosophila''': Ftz-F1 

*Mutants to identify regulated genes 
**nhr-25 is essential, therefore we have to knockdown the gene as opposed to knocking it out 
**transiently knocked down by RNAi- molting and vulval defects 

**nhr-25(ku217) mutant- molting and vulval defect 
***leucine to proline point mutation in the D 
NA biding domain 
***mutant has reduced DNA binding 

*identification of nhr-25 regulated genes 
**sequencing library consists of 10 worms from roughly the same developmental stage 

'''Pairwise COmparisons:'''
*WT vs Mutant 
*WT vs. Knockdown 
*Mutant vs. Knockdown 

*Narrow down list using adjusted p-value 
*Rank gene list based on fold change (largest to smallest) 
*Knockdown- moint mutation, mRNA is still in tact
*RNAi- binds to mRNA and mRNA is destroyed; no protein is created

Ky Roland

2018-03-20T13:42:33Z

Kyroland:

*25 and 24 = paired reads, Male mouse 2078, C201R 
'''23 and 22 = paired reads, Female mouse 2077, WT''' 
21 and 20 = paired reads, Male mouse 2076, WT 
19 and 18 = paired reads, Male mouse 2075, WT 
17 and 16 = paired reads, Male mouse 2074, WT 
*15 and 14 = paired reads, Male mouse 2073, C201R 
*'''13 and 12 = paired reads, Female mouse 2072, C201R''' 
*'''11 and 10 = paired reads, Female mouse 2071, C201R''' 
*'''9 and 8 = paired reads, Female mouse 2070, C201R''' 
'''7 and 6 = paired reads, Female mouse 2081, WT''' 
'''5 and 4 = paired reads, Female mouse 2080, WT''' 
*3 and 2 = paired reads, Male mouse 2079, C201R 

'''DESeq2- Comparison of WT vs. Mutant Females: Named-Types''' 
*Mutant will be in numerator, and WT will be int he denominator; a number > 1 will mean that there is an overexertion in the wild type.
*We should not expect differential expression because the papers showed no significant differential mRNA expression between the mutant and wild type.

'''Significant Genes:''' 
*'''Sphk2'''- [http://www.genecards.org/cgi-bin/carddisp.pl?gene=Sphk2 Gene Cards ] 
** Bears a relationship to Polr2a, another gene included in our list, but not significant 

Ontological Discovery Environment ID- What GeneWeaver uses 
*A common entity that gene species can map to in geneweaver 
*to develop sets that you can match 

Types of functional Genomics Data:

Mapping Data 
* QTL Positional Candidates 
*GWAS Candidate 

Expression Data: From NIF 
*Drug Related Genes Database- from literature 
*Allen Brain Atlas- Mouse 
** ensure localized expression 

View similar gene sets 
Hierarchical similarity graph- 
*Top nodes are the most highly connected nodes, with the most highly connected gene being red 

'''How to use GeneWeaver:''' 
[http://www.beta.geneweaver.org GeneWeaver Beta] 
* Search a key term and data related to your search term will appear ranked by relevance 
*You can use boolean queries to augment your search results to be more or less inclusive 

HiSim- a sort of multiway venn diagram without overlapping results of high level intersections of gene sets 
*Shows various combinations of intersecting gene sets 
*Emphasizes a relationship between the sets 

Gene Set Graph Tool 
*Different way of looking at data comparisons made in HiSim, but more useful for emphasizing the genes 

Jacard Similarity- 
View pairwise similarities and overlap 

no method for two different score types- can upload gene sets twice using different score types and then threshold them in analysis 

Might have to convert Gene identifiers if they are mixed in the gene set 

'''Gene Weaver Tips:''' 
*Use Gene ID's as the gene identifier 
----

== '''Transcription Factors''' ==
Start with Gene search in [https://www.ncbi.nlm.nih.gov/gene/2516 NCBI]. 

Where is this expressed? (all 4 data sets) 

Human conditions when mutated? 

How many proteins does NR5A1 interact with? 

What GO terms are associated with NR5A1? 

'''nhr-25: Model Transcription Factor''' 
*Background 
**an important developmental regulator 
**known phenotypes and tools 
**mammalian orthologs 

*NHR-25 
**transcription factor of large family: nuclear hormone receptors 
**expressed in seam and vulval cells throughout development 
**'''human orthologs''': SF-1 and LRH-1; and '''Drosophila''': Ftz-F1 

*Mutants to identify regulated genes 
**nhr-25 is essential, therefore we have to knockdown the gene as opposed to knocking it out 
**transiently knocked down by RNAi- molting and vulval defects 

**nhr-25(ku217) mutant- molting and vulval defect 
***leucine to proline point mutation in the D 
NA biding domain 
***mutant has reduced DNA binding 

*identification of nhr-25 regulated genes 
**sequencing library consists of 10 worms from roughly the same developmental stage 

'''Pairwise COmparisons:'''
*WT vs Mutant 
*WT vs. Knockdown 
*Mutant vs. Knockdown 

*Narrow down list using adjusted p-value 
*Rank gene list based on fold change (largest to smallest)

Ky Roland

2018-03-20T13:40:16Z

Kyroland: /* Transcription Factors */

*25 and 24 = paired reads, Male mouse 2078, C201R 
'''23 and 22 = paired reads, Female mouse 2077, WT''' 
21 and 20 = paired reads, Male mouse 2076, WT 
19 and 18 = paired reads, Male mouse 2075, WT 
17 and 16 = paired reads, Male mouse 2074, WT 
*15 and 14 = paired reads, Male mouse 2073, C201R 
*'''13 and 12 = paired reads, Female mouse 2072, C201R''' 
*'''11 and 10 = paired reads, Female mouse 2071, C201R''' 
*'''9 and 8 = paired reads, Female mouse 2070, C201R''' 
'''7 and 6 = paired reads, Female mouse 2081, WT''' 
'''5 and 4 = paired reads, Female mouse 2080, WT''' 
*3 and 2 = paired reads, Male mouse 2079, C201R 

'''DESeq2- Comparison of WT vs. Mutant Females: Named-Types''' 
*Mutant will be in numerator, and WT will be int he denominator; a number > 1 will mean that there is an overexertion in the wild type.
*We should not expect differential expression because the papers showed no significant differential mRNA expression between the mutant and wild type.

'''Significant Genes:''' 
*'''Sphk2'''- [http://www.genecards.org/cgi-bin/carddisp.pl?gene=Sphk2 Gene Cards ] 
** Bears a relationship to Polr2a, another gene included in our list, but not significant 

Ontological Discovery Environment ID- What GeneWeaver uses 
*A common entity that gene species can map to in geneweaver 
*to develop sets that you can match 

Types of functional Genomics Data:

Mapping Data 
* QTL Positional Candidates 
*GWAS Candidate 

Expression Data: From NIF 
*Drug Related Genes Database- from literature 
*Allen Brain Atlas- Mouse 
** ensure localized expression 

View similar gene sets 
Hierarchical similarity graph- 
*Top nodes are the most highly connected nodes, with the most highly connected gene being red 

'''How to use GeneWeaver:''' 
[http://www.beta.geneweaver.org GeneWeaver Beta] 
* Search a key term and data related to your search term will appear ranked by relevance 
*You can use boolean queries to augment your search results to be more or less inclusive 

HiSim- a sort of multiway venn diagram without overlapping results of high level intersections of gene sets 
*Shows various combinations of intersecting gene sets 
*Emphasizes a relationship between the sets 

Gene Set Graph Tool 
*Different way of looking at data comparisons made in HiSim, but more useful for emphasizing the genes 

Jacard Similarity- 
View pairwise similarities and overlap 

no method for two different score types- can upload gene sets twice using different score types and then threshold them in analysis 

Might have to convert Gene identifiers if they are mixed in the gene set 

'''Gene Weaver Tips:''' 
*Use Gene ID's as the gene identifier 
----

== '''Transcription Factors''' ==
Start with Gene search in [https://www.ncbi.nlm.nih.gov/gene/2516 NCBI]. 

Where is this expressed? (all 4 data sets) 

Human conditions when mutated? 

How many proteins does NR5A1 interact with? 

What GO terms are associated with NR5A1? 

'''nhr-25: Model Transcription Factor''' 
*Background 
**an important developmental regulator 
**known phenotypes and tools 
**mammalian orthologs 

*NHR-25 
**transcription factor of large family: nuclear hormone receptors 
**expressed in seam and vulval cells throughout development 
**'''human orthologs''': SF-1 and LRH-1; and '''Drosophila''': Ftz-F1 

*Mutants to identify regulated genes 
**nhr-25 is essential, therefore we have to knockdown the gene as opposed to knocking it out 
**transiently knocked down by RNAi- molting and vulval defects 

**nhr-25(ku217) mutant- molting and vulval defect 
***leucine to proline point mutation in the D 
NA biding domain 
***mutant has reduced DNA binding 

*identification of nhr-25 regulated genes 
**sequencing library consists of 10 worms from roughly the same developmental stage 

'''Pairwise COmparisons:'''
*WT vs Mutant 
*WT vs. Knockdown 
*Mutant vs. Knockdown

Ky Roland

2018-03-15T14:32:53Z

Kyroland: /* Transcription Factors */

*25 and 24 = paired reads, Male mouse 2078, C201R 
'''23 and 22 = paired reads, Female mouse 2077, WT''' 
21 and 20 = paired reads, Male mouse 2076, WT 
19 and 18 = paired reads, Male mouse 2075, WT 
17 and 16 = paired reads, Male mouse 2074, WT 
*15 and 14 = paired reads, Male mouse 2073, C201R 
*'''13 and 12 = paired reads, Female mouse 2072, C201R''' 
*'''11 and 10 = paired reads, Female mouse 2071, C201R''' 
*'''9 and 8 = paired reads, Female mouse 2070, C201R''' 
'''7 and 6 = paired reads, Female mouse 2081, WT''' 
'''5 and 4 = paired reads, Female mouse 2080, WT''' 
*3 and 2 = paired reads, Male mouse 2079, C201R 

'''DESeq2- Comparison of WT vs. Mutant Females: Named-Types''' 
*Mutant will be in numerator, and WT will be int he denominator; a number > 1 will mean that there is an overexertion in the wild type.
*We should not expect differential expression because the papers showed no significant differential mRNA expression between the mutant and wild type.

'''Significant Genes:''' 
*'''Sphk2'''- [http://www.genecards.org/cgi-bin/carddisp.pl?gene=Sphk2 Gene Cards ] 
** Bears a relationship to Polr2a, another gene included in our list, but not significant 

Ontological Discovery Environment ID- What GeneWeaver uses 
*A common entity that gene species can map to in geneweaver 
*to develop sets that you can match 

Types of functional Genomics Data:

Mapping Data 
* QTL Positional Candidates 
*GWAS Candidate 

Expression Data: From NIF 
*Drug Related Genes Database- from literature 
*Allen Brain Atlas- Mouse 
** ensure localized expression 

View similar gene sets 
Hierarchical similarity graph- 
*Top nodes are the most highly connected nodes, with the most highly connected gene being red 

'''How to use GeneWeaver:''' 
[http://www.beta.geneweaver.org GeneWeaver Beta] 
* Search a key term and data related to your search term will appear ranked by relevance 
*You can use boolean queries to augment your search results to be more or less inclusive 

HiSim- a sort of multiway venn diagram without overlapping results of high level intersections of gene sets 
*Shows various combinations of intersecting gene sets 
*Emphasizes a relationship between the sets 

Gene Set Graph Tool 
*Different way of looking at data comparisons made in HiSim, but more useful for emphasizing the genes 

Jacard Similarity- 
View pairwise similarities and overlap 

no method for two different score types- can upload gene sets twice using different score types and then threshold them in analysis 

Might have to convert Gene identifiers if they are mixed in the gene set 

'''Gene Weaver Tips:''' 
*Use Gene ID's as the gene identifier 
----

== '''Transcription Factors''' ==
Start with Gene search in [https://www.ncbi.nlm.nih.gov/gene/2516 NCBI]. 

Where is this expressed? (all 4 data sets) 

Human conditions when mutated? 

How many proteins does NR5A1 interact with? 

What GO terms are associated with NR5A1? 

'''nhr-25: Model Transcription Factor''' 
*Background 
**an important developmental regulator 
**known phenotypes and tools 
**mammalian orthologs 

*NHR-25 
**transcription factor of large family: nuclear hormone receptors 
**expressed in seam and vulval cells throughout development 
**'''human orthologs''': SF-1 and LRH-1; and '''Drosophila''': Ftz-F1 

*Mutants to identify regulated genes 
**nhr-25 is essential, therefore we have to knockdown the gene as opposed to knocking it out 
**transiently knocked down by RNAi- molting and vulval defects 
**nhr-25(ku217) mutant- molting and vulval defect 
***leucine to proline point mutation in the D 
NA biding domain 
***mutant has reduced DNA binding 

*identification of nhr-25 regulated genes 
**sequencing library consists of 10 worms from roughly the same developmental stage

Ky Roland

2018-03-15T13:44:47Z

Kyroland: /* Transcription Factors */

Ky Roland

2018-03-15T13:44:36Z

Kyroland: /* Transcription Factors */

Ky Roland

2018-03-15T13:43:57Z

Kyroland: /* Transcription Factors */

Ky Roland

2018-03-15T13:43:39Z

Kyroland: /* Transcription Factors */

Ky Roland

2018-03-13T14:56:30Z

Kyroland: /* Transcription Factors */

Ky Roland

2018-03-13T14:56:11Z

Kyroland:

Ky Roland

2018-02-20T15:31:00Z

Kyroland:

Ky Roland

2018-02-15T15:49:57Z

Kyroland:

Ky Roland

2018-02-15T15:40:56Z

Kyroland:

Ky Roland

2018-02-08T15:57:11Z

Kyroland:

Ky Roland

2018-02-08T15:25:50Z

Kyroland:

Ky Roland

2018-02-08T15:10:44Z

Kyroland:

Ky Roland

2018-02-08T15:09:03Z

Kyroland:

Ky Roland

2018-02-08T14:45:11Z

Kyroland:

Ky Roland

2018-02-08T14:45:03Z

Kyroland:

Ky Roland

2018-02-08T14:43:55Z

Kyroland:

Ky Roland

2018-02-06T15:59:18Z

Kyroland:

25 and 24 = paired reads, Male mouse 2078, C201R 
'''23 and 22 = paired reads, Female mouse 2077, WT''' 
21 and 20 = paired reads, Male mouse 2076, WT 
19 and 18 = paired reads, Male mouse 2075, WT 
17 and 16 = paired reads, Male mouse 2074, WT 
15 and 14 = paired reads, Male mouse 2073, C201R 
'''13 and 12 = paired reads, Female mouse 2072, C201R''' 
'''11 and 10 = paired reads, Female mouse 2071, C201R''' 
'''9 and 8 = paired reads, Female mouse 2070, C201R''' 
'''7 and 6 = paired reads, Female mouse 2081, WT''' 
'''5 and 4 = paired reads, Female mouse 2080, WT''' 
3 and 2 = paired reads, Male mouse 2079, C201R 

'''DESeq2- Comparison of WT vs. Mutant Females:''' 
*Mutant will be in numerator, and WT will be int he denominator; a number > 1 will mean that there is an overexertion in the wild type.
*We should not expect differential expression because the papers showed no significant differential mRNA expression between the mutant and wild type.

Ky Roland

2018-02-06T15:59:02Z

Kyroland:

25 and 24 = paired reads, Male mouse 2078, C201R 
'''23 and 22 = paired reads, Female mouse 2077, WT''' 
21 and 20 = paired reads, Male mouse 2076, WT 
19 and 18 = paired reads, Male mouse 2075, WT 
17 and 16 = paired reads, Male mouse 2074, WT 
15 and 14 = paired reads, Male mouse 2073, C201R 
'''13 and 12 = paired reads, Female mouse 2072, C201R''' 
'''11 and 10 = paired reads, Female mouse 2071, C201R''' 
'''9 and 8 = paired reads, Female mouse 2070, C201R''' 
'''7 and 6 = paired reads, Female mouse 2081, WT''' 
'''5 and 4 = paired reads, Female mouse 2080, WT''' 
3 and 2 = paired reads, Male mouse 2079, C201R 

'''DESeq2'''- Comparison of WT vs. Mutant Females 
*Mutant will be in numerator, and WT will be int he denominator; a number > 1 will mean that there is an overexertion in the wild type.
*We should not expect differential expression because the papers showed no significant differential mRNA expression between the mutant and wild type.

Ky Roland

2018-02-06T15:58:43Z

Kyroland:

25 and 24 = paired reads, Male mouse 2078, C201R 
'''23 and 22 = paired reads, Female mouse 2077, WT''' 
21 and 20 = paired reads, Male mouse 2076, WT 
19 and 18 = paired reads, Male mouse 2075, WT 
17 and 16 = paired reads, Male mouse 2074, WT 
15 and 14 = paired reads, Male mouse 2073, C201R 
'''13 and 12 = paired reads, Female mouse 2072, C201R''' 
'''11 and 10 = paired reads, Female mouse 2071, C201R''' 
'''9 and 8 = paired reads, Female mouse 2070, C201R''' 
'''7 and 6 = paired reads, Female mouse 2081, WT''' 
'''5 and 4 = paired reads, Female mouse 2080, WT''' 
3 and 2 = paired reads, Male mouse 2079, C201R 

DESeq2- Comparison of WT vs. Mutant Females 
*Mutant will be in numerator, and WT will be int he denominator; a number > 1 will mean that there is an overexertion in the wild type.
*We should not expect differential expression because the papers showed no significant differential mRNA expression between the mutant and wild type.

Ky Roland

2018-02-06T15:51:08Z

Kyroland:

Ky Roland

2018-02-06T15:50:54Z

Kyroland:

Ky Roland

2018-02-06T15:50:14Z

Kyroland:

Ky Roland

2018-02-06T15:49:41Z

Kyroland: Created page with "25 and 24 = paired reads, Male mouse 2078, C201R '''23 and 22 = paired reads, Female mouse 2077, WT''' 21 and 20 = paired reads, Male mouse 2076, WT 19 and 18 = paired reads,..."

25 and 24 = paired reads, Male mouse 2078, C201R
'''23 and 22 = paired reads, Female mouse 2077, WT'''
21 and 20 = paired reads, Male mouse 2076, WT
19 and 18 = paired reads, Male mouse 2075, WT
17 and 16 = paired reads, Male mouse 2074, WT
15 and 14 = paired reads, Male mouse 2073, C201R
'''13 and 12 = paired reads, Female mouse 2072, C201R
11 and 10 = paired reads, Female mouse 2071, C201R'''
'''9 and 8 = paired reads, Female mouse 2070, C201R
7 and 6 = paired reads, Female mouse 2081, WT
5 and 4 = paired reads, Female mouse 2080, WT'''
3 and 2 = paired reads, Male mouse 2079, C201R

What does Timmomatic do?

2018-02-06T15:30:54Z

Kyroland:

'''Trimmomatic''' is a program that remove adapters from FastQ data so that this tagging information is not included in future data analysis and mapping. 

Running FastQ data through trimmomatic increases quality control scores by removing lower quality reads form the data. 

You can run trimmomatic on single reads or read pairs ('''Single end mode or Paired end mode'''). Paired mode uses data from paired reads to improve the likelihood of finding adapters and PCR primer fragments within the data by using forward and reverse reads of your data.

[http://www.usadellab.org/cms/uploads/supplementary/Trimmomatic/TrimmomaticManual_V0.32.pdf Source of Trimmomatic Data]

What does Timmomatic do?

2018-02-06T15:29:37Z

Kyroland:

'''Trimmomatic''' is a program that remove adapters from FastQ data so that this tagging information is not included in future data analysis and mapping. 

Running FastQ data through trimmomatic increases quality control scores by removing lower quality reads form the data. 

You can run trimmomatic on single reads or read pairs (Single mode or Paired mode). Paired mode uses data from paired reads to improve the likelihood of finding adapters and PCR primer fragments within the data by using forward and reverse reads of your data.

[http://www.usadellab.org/cms/uploads/supplementary/Trimmomatic/TrimmomaticManual_V0.32.pdf Source of Trimmomatic Data]

What does Timmomatic do?

2018-02-06T15:28:38Z

Kyroland:

'''Trimmomatic''' is a program that remove adapters from FastQ data so that this tagging information is not included in future data analysis and mapping. 
Running FastQ data through trimmomatic increases quality control scores by removing lower quality reads form the data. 
You can run trimmomatic on single reads or read pairs (Single mode or Paired mode). Paired mode uses data from paired reads to improve the likelihood of finding adapters and PCR primer fragments within the data by using forward and reverse reads of your data.

[http://www.usadellab.org/cms/uploads/supplementary/Trimmomatic/TrimmomaticManual_V0.32.pdf Source of Trimmomatic Data]

What does Timmomatic do?

2018-02-06T15:26:06Z

Kyroland:

'''Trimmomatic''' is a program that remove adapters from FastQ data so that this tagging information is not included in future data analysis and mapping. <brb>
Running FastQ data through trimmomatic increases quality control scores by removing lower quality reads form the data. <brb>
You can run trimmomatic on single reads or read pairs (Single mode or Paired mode). Paired mode uses data from paired reads to improve the likelihood of finding adapters and PCR primer fragments within the data by using forward and reverse reads of your data.

[http://www.usadellab.org/cms/uploads/supplementary/Trimmomatic/TrimmomaticManual_V0.32.pdf Source of Trimmomatic Data]

What does Timmomatic do?

2018-02-06T15:18:58Z

Kyroland:

Trimmomatic is a program that remove adapters from FastQ data so that this tagging information is not included in future data analysis and mapping. Running FastQ data through trimmomatic increases the quality control scores by removing lower quality reads form the data. You can run trimmomatic on single reads or read pairs (Single mode or Paired mode). Paired mode uses data from paired reads to improve the likelihood of finding adapters and PCR primer fragments within the data.

[http://www.usadellab.org/cms/uploads/supplementary/Trimmomatic/TrimmomaticManual_V0.32.pdf Source of Trimmomatic Data]

What does Timmomatic do?

2018-02-06T15:06:37Z

Kyroland:

Trimmomatic is a program that remove adapters from FastQ data so that this tagging information is not included in future data analysis. Running FastQ data through trimmomatic increases the quality control scores.

[http://www.usadellab.org/cms/uploads/supplementary/Trimmomatic/TrimmomaticManual_V0.32.pdf Source of Trimmomatic Data]

What does Timmomatic do?

2018-02-06T15:05:09Z

Kyroland:

Trimmomatic is a program that remove adapters from FastQ data so that this tagging information is not included in future data analysis. Running a FastQ data through trimmomatic increases it's quality control read.

[http://www.usadellab.org/cms/uploads/supplementary/Trimmomatic/TrimmomaticManual_V0.32.pdf Source of Trimmomatic Data]

What does Timmomatic do?

2018-02-06T15:04:43Z

Kyroland:

What does Timmomatic do?

2018-02-06T14:59:21Z

Kyroland: Created page with "Trimmomatic is a program that remove adapters from FASTQ data so that this tagging information is not included in future data analysis. The FASTQC [http://www.usadellab.or..."

Trimmomatic is a program that remove adapters from FASTQ data so that this tagging information is not included in future data analysis. The FASTQC

[http://www.usadellab.org/cms/uploads/supplementary/Trimmomatic/TrimmomaticManual_V0.32.pdf]

Stephanie and Ky

2018-02-06T14:46:23Z

Kyroland:

[[What does Timmomatic do?]]

[[How is it connected to FastQC?]]