Samples were submitted to the Broad Institute’s Genomics Platform for transcriptome library construction following the dUTP protocol30 and Illumina sequencing. 5 micrograms of total RNA as measured by RiboGreen at a concentration of 50 nanogram/microliter with RNA Integrity Number (RIN) score of 5 or better were submitted for cDNA library construction. RIN score affects the fragment lengths of RNA inserts for library construction, and therefore we batched samples according to RIN scores so that library pools would have uniform insert sizes. 582 subjects in 6 batches/plates containing up to 92 samples, were processed using the dUTP method, barcoded and pooled for sequencing. Subsequently, 52 samples in a single batch were processed using the newer Illumina TruSeq method modified by The Broad Institute Genomics Platform to be strand specific and to use larger insert sizes. The resulting library closely resembles the library obtained by the dUTP method. The Truseq method uses only 250 nanograms of RNA input. Sequencing was carried out using the Illumina HiSeq2000 with 101 bp paired end reads for a targeted coverage of 50M paired reads.
