RNA-Seq library preparation and sequencing: Site 2
Total RNA integrity was determined using Agilent Bioanalyzer or 4200 Tapestation. ERCC synthetic spike-in 1 or 2 (Thermo Fisher Scientific Inc., Waltham, Massachusetts) was added to each total RNA sample and processed in parallel. Library preparation was performed with 1 ug, of total RNA with a Bioanalyzer RIN score greater than 8.0. Ribosomal RNA was removed by poly-A selection using Oligo-dT beads (mRNA Direct kit, Life Technologies. In cases, where samples had less than 1ug of total RNA, the entire available amount was used. mRNA was then fragmented in buffer containing 40 mM Tris Acetate pH 8.2, 100 mM Potassium Acetate, and 30 mM Magnesium Acetate and heated to 94 degrees for 150 seconds. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer’s instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3’ ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 14 cycles using primers incorporating unique index tags. Fragments were sequenced on an Illumina HiSeq 3000 using single-end reads extending 100 bases to an average of 35 M reads per sample.