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Protocols
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mRNA Sequencing Sample Preparation Guide
(Vincent J. Coates Genomic Sequencing Lab, UC Berkeley)
This protocol explains how to convert total RNA into a library of template molecules suitable for high throughput DNA sequencing for subsequent cluster generation. The first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T oligo-attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. Then the cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNaseH. These cDNA fragments then go through an end repair process, the addition of a single ‘A’ base, and then ligation of the adapters. These products are then purified and enriched with PCR to create the final cDNA library.
http://qb3.berkeley.edu/gsl/Protocols_files/mRNA-S...
Added: Sat Mar 27 2010, Hits: 1199, Reviews: 0 Write review -
Small RNA Sequencing Sample Preparation Guide
(Vincent J. Coates Genomic Sequencing Lab, UC Berkeley)
This protocol explains how to prepare small RNA libraries using the alternative v1.5 for subsequent sequencing during cluster generation.
http://qb3.berkeley.edu/gsl/Protocols_files/SmallR...
Added: Sat Mar 27 2010, Hits: 604, Reviews: 0 Write review -
Tech Summary: Illumina's Solexa Sequencing Technology
(SEQanswers)
An overview of Solexa's sequencing-by-synthesis chemistry. The sample prep methods used differ slightly from that used in ABI's SOLiD system, but the basic goals are the same: generate large numbers of unique "polonies" (polymerase generated colonies) that can be simultaneously sequenced. These parallel reactions occur on the surface of a "flow cell" (basically a water-tight microscope slide) which provides a large surface area for many thousands of parallel chemical reactions.
http://seqanswers.com/forums/showthread.php?t=21
Added: Sat Mar 27 2010, Hits: 829, Reviews: 0 Write review Cached