Poly-T capture beads were used to isolate mRNA from 10 ug of total RNA. First strand cDNA was generated using random hexamer-primed reverse transcription, and subsequently used to generate second strand cDNA using RNase H and DNA polymerase. Sequencing adapters were ligated using the Illumina Genomic DNA sample prep kit. Fragments ∼200 bp long were isolated by gel electrophoresis, amplified by 16 cycles of PCR, and sequenced on the Illumina Genome Analyzer, as described40 .
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Transcriptome Library Preparation Protocol
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Other organizations : Massachusetts Institute of Technology, Illumina (United States), Whitehead Institute for Biomedical Research, National Center for Genome Resources
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- Use of Poly-T capture beads to isolate mRNA from 10 ug of total RNA
- Generation of first strand cDNA using random hexamer-primed reverse transcription
- Generation of second strand cDNA using RNase H and DNA polymerase
- Ligation of sequencing adapters using the Illumina Genomic DNA sample prep kit
- Isolation of fragments ~200 bp long by gel electrophoresis
- Amplification of fragments by 16 cycles of PCR
- Sequencing on the Illumina Genome Analyzer
- Not explicitly mentioned
- Not explicitly mentioned
- No positive or negative controls specified
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