RNA-Seq analysis of HS1907
Total RNA from 12 Normal tissue samples was purified using Qiagen’s Rneasy kit with on-column DNase digestion. Ribosomal RNA was removed using Invitrogen’s RiboMinus kit. rRNA contamination (as estimated by Agilent analysis) ranged from undetectable to 4.5%. 1st and 2nd strand cDNA was synthesized from RiboMinus–purified RNA using random hexamers and Invitrogen’s Superscript II cDNA kit. PAGE-purified 200-300 bp size fraction DNAs were polished, A-tailed, ligated to PE adapters and PCR amplified using primer 1.0 and one of 12 indexed primers (IDX2-000001 through IDX2-000012). Relative yield of the 320-420 bp product was estimated using densitometry (with samples loaded on a PAGE gel). Equimolar amounts of the 12 samples were pooled and the 320-420bp product was PAGE-purified, precipitated with Glycogen (Ambion) carrier and resuspended in 11.5 ul EB (1.5 ul used for Nanodrop)