GSM4932382: 2011-325 RNA84 [control]; Homo sapiens; RNA-Seq
RNA libraries were prepared using TruSeq RNA  Sample Prep Kit v2 (Illumina, San Diego, CA). Briefly, poly(A)  mRNA was purified from total RNA using oligo(dT) magnetic  beads, fragmented at 95 °C for 8 min, eluted from the beads, and  primed for first-strand cDNA synthesis using SuperScript III  reverse transcriptase and random primers (Invitrogen). Second-  strand cDNA synthesis was performed using DNA polymerase  I and RNase H, and double-stranded cDNA was purified  using a single AMPure XP bead cleanup step (Agencourt,  Danvers, MA). The cDNA ends were repaired and phosphorylated  using Klenow fragment, T4 polymerase, and T4 polynucleotide  kinase, followed by a single AMPure XP bead cleanup  step. The blunt-ended cDNAs were modified to include a single  3 -adenylate residue using Klenow exo  (3  to 5  exo ), and  paired-end DNA adaptors (Illumina) with a single T base overhang  at the 3 -end were ligated to the A-tailed cDNA population.  Unique indexes, included in the standard TruSeq kits (12-  Set A and 12-Set B), were incorporated at the adaptor ligation  step for multiplex sample loading on the flow cells. The resulting  constructs were purified by two consecutive AMPure XP  bead cleanup steps and enriched by 12 cycles of PCR using  primers included in TruSeq RNA Sample Prep Kit v2. Libraries  were loaded onto paired-end flow cells at concentrations of  8–10 pM to generate cluster densities of 700,000/mm2 using  cBot and cBot Paired-end Cluster Kit v3 (Illumina) following  the manufacturer's standard protocol. The flow cells were  sequenced as 51   2 paired-end reads on an Illumina HiSeq  4000 system using TruSeq SBS Sequencing Kit v3 and SCS  v1.4.8 data collection software.