high throughput sequence alignment protocol
Raw sequence reads were trimmed to 39 bp due to abundant presence of adapter sequences and mapped against the standard hg19 build of the human reference genome with BWA 0.5.9 (http://bio-bwa.sourceforge.net) with default parameters for paired-end reads. Only read 1 was used for further analyses after trimming given that read 2 would overlap read 1 in many cases. Uniquely mapped reads with a mapping quality >= 10 were merged from the different sequenced libraries originating from the same sample.
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 50 bp). The libraries were multiplexed as pools of three individuals, and sequenced once (short libraries) or twice (long libraries).
nucleic acid extraction protocol
Nuclei were isolated and nucleotides washed off at 4 degrees C, leaving RNA polymerases engaged in transcription bound to DNA. 5M nuclei per sample were then used for letting the polymerases run-on for ~100 nts using Br-UTP, alpha-P32-labeled CTP for tracking the nascent RNA through the experiment and sarkosyl for blocking new transcription initiation events. RNA was isolated and hydrolyzed. Subsequently, the nascent RNA was pulled down with agarose beads carrying antibodies for Br-UTP. 5' and 3' adapters were then ligated to nascent RNA, fulfilling another round of immuno-enrichment after each step. Finally, the nascent RNA library was converted to cDNA, PCR amplified and purified, yielding 2 libraries per sample. Given the way they were purified from the agarose gel, one library is of longer insert size (termed long) than the other (termed short).
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.