high throughput sequence alignment protocol
Raw sequence reads were mapped against the standard hg19 build of the human reference genome using BWA 0.5.9 (http://bio-bwa.sourceforge.net) with default parameters for paired-end reads. Properly paired uniquely mapping reads with a mapping quality >= 10 were kept for further analyses.
nucleic acid sequencing protocol
Sequencing was performed on the Illumina HiSeq 2000 platform using TruSeq PE Cluster Kit v3 (cluster generation) and TruSeq SBS Kit v3 (paired-end sequencing, read length 49 bp). Samples were multiplexed and sequenced either six or twelve samples per lane (Pilot2 and Pilot1 samples, respectively).
nucleic acid extraction protocol
Total RNA was extracted from flash frozen cell pellets using the TRIzol Reagent (Invitrogen) according to manufacturer's instructions. No DNAse treatment of the RNA samples was performed. Total RNA quality was assessed with Bioanalyzer RNA 6000 Nano Kit (Agilent) and RNA quantity was measured with Qubit 2.0 (Invitrogen) RNA Broad range kit according to manufacturer's instructions.
Lymphoblastoid cell lines were obtained from Coriell (http://ccr.coriell.org) and cultured in RPMI-1640 medium (Lonza) with 10% fetal calf serun (FCS). Frozen cells were thawed and transferred to T25 flasks containing 15 ml of medium. At a density of 0.3 x 10^6 cells/m cells were transferred to TubeSpin Bioreactor 50 tubes (TPP) in 5 ml of same medium containing 10% FCS and 0.1% Pluronic F-68 (Sigma-Aldrich). The cultures were agitated at 180 rpm with 5% CO2 and 85% humidity. When the cell density reached 2-3 x 10^6 cells/ml, the culture was diluted to 0.3 x 10^6 cells/ml and transferred to a 250-ml glass bottle. The culture was agitated at 110 rpm with 5% CO2 but no humidity. Eventually, the cells were scaled-up serially in 500-ml, 1-L, and 5-L glass bottles filled to a maximum of 40% of the nominal volume.
nucleic acid library construction protocol
Sequencing libraries were prepared with TruSeq RNA Sample Prep Kit v2 high-throughput protocol (Illumina) according to manufacturer's instructions.