Illumina HiSeq 2000 paired end sequencing; Coordinated effects of sequence variation on DNA binding, chromatin structure, and transcription.
Coordinated effects of sequence variation on DNA binding, chromatin structure, and transcription.
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. 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). Sequencing libraries were prepared with TruSeq RNA Sample Prep Kit v2 high-throughput protocol (Illumina) according to manufacturer's instructions.
Experimental Factor: individual
NA12891