DM Notes 1.12.16 - Revision history

Dymaghini at 18:39, 11 February 2016

2016-02-11T18:39:51Z

Dymaghini at 18:39, 11 February 2016

2016-02-11T18:39:38Z

Dymaghini at 20:41, 27 January 2016

2016-01-27T20:41:13Z

Dymaghini: Created page with "RNAseq protocol: Flash freeze tissue from six snakes, two organs per snake RNA range: 100 ng to 10 micrograms --> how do we know that the correct portion of ti..."

2016-01-14T18:35:22Z

Created page with "RNAseq protocol: Flash freeze tissue from six snakes, two organs per snake RNA range: 100 ng to 10 micrograms --> how do we know that the correct portion of ti..."

New page

RNAseq protocol:
Flash freeze tissue from six snakes, two organs per snake
RNA range: 100 ng to 10 micrograms
--> how do we know that the correct portion of tissue was sampled?
Convert RNA to mRNA using polyT oligonucleotides attached to beads, such that mRNA base pairs with beads
Convert mRNA to cDNA with RT (reverse transcriptase, uses RNA as a template to make DNA) and dNTP)

-RNA fragmentation, cDNA synthesis, and clean-up
high throughput sequencing has only ~75bp of accurate reads
fragmentation takes one long mRNA and cuts it into similarly sized fragments so that there are more edges to read from
How to prime every RNA simultaneously? Generate every possible hexamer for the primers (4^6 different combinations)
Product:
Look at images in pptx
Second band of images: gel purified RNA
Third band of images: reamplified (considered good cDNA library for RNAseq)

← Older revision		Revision as of 18:39, 11 February 2016
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	Second band of images: gel purified RNA		Second band of images: gel purified RNA
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		+	Third band of images: reamplified (considered good cDNA library for RNAseq)

	Back to home [[Dylan Maghini]]		Back to home [[Dylan Maghini]]
−
−	~~Third band of images: reamplified (considered good cDNA library for RNAseq)~~

← Older revision		Revision as of 18:39, 11 February 2016
Line 24:		Line 24:

	Second band of images: gel purified RNA		Second band of images: gel purified RNA
		+
		+	Back to home [[Dylan Maghini]]

	Third band of images: reamplified (considered good cDNA library for RNAseq)		Third band of images: reamplified (considered good cDNA library for RNAseq)

@@ Line 1: / Line 1: @@
-RNAseq protocol:
+RNAseq protocol
-     -RNA fragmentation, cDNA synthesis, and clean-up
+Flash freeze tissue from six snakes, two organs per snake
-        high throughput sequencing has only ~75bp of accurate reads
-        fragmentation takes one long mRNA and cuts it into similarly sized fragments so that there are more edges to read from
+RNA range: 100 ng to 10 micrograms
-        How to prime every RNA simultaneously? Generate every possible hexamer for the primers (4^6 different combinations)
-    Product:
+--> how do we know that the correct portion of tissue was sampled?
-    Look at images in pptx
-    Second band of images: gel purified RNA
+Convert RNA to mRNA using polyT oligonucleotides attached to beads, such that mRNA base pairs with beads
-    Third band of images: reamplified (considered good cDNA library for RNAseq)