Sequencing libraries were generated with the Illumina TruSeq RNA Sample Preparation Kit. Briefly, RNA molecules were fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA synthesis using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments were end-repaired using T4 DNA polymerase, Klenow polymerase and T4 polynucleotide kinase. The resulting blunt-ended fragments were A-tailed using a 3′–5′ exonuclease-deficient Klenow fragment and ligated to Illumina adaptor oligonucleotides in a ‘TA’ ligation. The ligation mixture was further size-selected by AMPure beads and enriched by PCR amplification following Illumina TruSeq DNA Sample Preparation protocol. The resulting library is attached and amplified on a flow-cell by cBot Cluster Generation System.
The sequencing was done with an Illumina HiSeq2000 sequencer. Multiplexing was used to pool 4 samples into one sequencing lane. After each sequencing run, the raw reads were pro-processed to filter out low quality reads and to remove the multiplexing barcode sequences. The dataset is available through the NCBI GEO database (accession number GSE111222).
The sequencing was done with an Illumina HiSeq2000 sequencer. Multiplexing was used to pool 4 samples into one sequencing lane. After each sequencing run, the raw reads were pro-processed to filter out low quality reads and to remove the multiplexing barcode sequences. The dataset is available through the NCBI GEO database (accession number GSE111222).