Illumina Genome Analyzer IIx 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. 2-L cell cultures at a density of 0.8_0.9 x 10^6 cells/ml in 5-L bottles were mounted on a shaker and agitated at 70 rpm at room temperature. Formaldehyde (Sigma-Aldrich) was slowly added to a final concentration of 0.8% and agitation was continued for 7 min. The fixation was quenched by addition of 2.5 M glycine (Rectolab) to a final concentration of 0.125 M, and the culture was agitated as before for 5 min. The cells were collected by centrifugation at 2000 rpm for 5 min at 4  degrees C and then washed 4 times with cold PBS. The last centrifugation step was performed in 50-ml centrifuge tubes, each containing 50 x 10^6 cells. The final cell pellets were flash frozen in liquid nitrogen and stored at -80  degrees C. [RPB2 and TFIIB]: ChIPs were carried out as described by Canella et al. 2010 (PMID: 20413673) with a few modifications. Chromatin extracted from 5 x 10^7 crosslinked cells was sonicated to an average size of 200-700 bp. Sheared chromatin was then immunoprecipitated with 7 ug per 10^7 cells of an anti-Rpb2 antibody or 7.5 ul per 10^7 cells of an anti-TFIIB antibody (see Schramm et al. 2000; PMID:11040218). Immunoprecipitated material was recovered with 2 mg per 10^7 cells of pre-blocked protein-A beads (GE Healthcare) and washed twice with dialysis buffer and three times with IP wash buffer. After reversal of crosslinking and DNA purification, 10 ng of ChIP DNA was used for ChIP-seq libraries preparation. ChIP libraries were prepared for sequencing with the Illumina ChIP-seq sample preparation kit (all pilot2 samples, except H3K27ac) and the TruSeq DNA sample prep kit (all pilot1 samples and pilot2 samples of H3K27ac), according to manufacturer's instructions. With the ChIP-seq kit, the number of PCR cycles used to amplify the libraries was either 18 (POLR2B) or 17 (all other assays). With the TruSeq kit, indexing adapters AD001-AD002 were used to index the samples, according to manufacturer's recommendations. ChIP DNA concentration was re-measured prior to library preparation. The starting amount of DNA was 6-10.5 ng (pilot2) or 2.5-10.5 ng (pilot1) per sample. Library quality and average fragment size was confirmed with Bioanalyzer 25-1000bp DNA analysis kit (Agilent).
Experimental Factor: individual
NA12776
Experimental Factor: population
HapMap CEU
Experimental Factor: immunoprecipitate
TFIIB