GSM2873885: Control replicate 2 [Nascent RNA-Seq]; Homo sapiens; RNA-Seq
Click-iT™ Nascent RNA Capture Kit (Invitrogen, cat#C10365) Nascent RNA capture and 1st strand cDNA synthesis were performed using the Click-iT Nascent RNA Capture Kit (Molecular Probes).  2nd strand cDNA synthesis was performed using Illumina Second Strand Mix, which incorporates dUTP to preserve strand data.  Full length ds cDNA was purified using AmpureXP beads (Beckman Coulter) and sheared using a Covaris S220 (Covaris Inc) to a final size of 150-200bp.  KAPA Hyper Prep (KAPA Biosystems) reagents were used to make final libraries following the kit instructions for end repair, A tailing, ligation of Illumina TruSeq Stranded RNA indexes, and PCR amplification.  Amplified final libraries were purified using AmpureXP beads, quantitated using qPCR (KAPA Biosystems), and pooled in an equimolar fashion.  Pooled libraries are loaded to an individual lane of a HiSeq High Output v3 flow cell, using an Illumina cBot, for 50cycle SR sequencing on a HiSeq2500 sequencer according to the manufacturer's recommended protocol (Illumina Inc.).