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. [H3K4me3, H3K27me3, H4K20me1, H3K27ac]: ChIP was carried out largely as suggested by O'Geen et al. 2006 (PMID:17140114), with modifications made to automatize the procedure. Briefly, cells were lysed by addition of cell lysis buffer, then nuclei were washed and subsequently lysed using nuclei lysis buffer. Chromatin was sheared with Covaris S220 sonicator. Sonication efficiency was assessed by running a sample of de-crosslinked DNA on a 1.5% agarose gel. Fragmented chromatin was diluted 10 fold (5 fold in case of H3K27ac IP) in ChIP dilution buffer and immunoprecipitated using respective antibodies. The immunoprecipitation assays were performed on Diagenode SX-8G IP-Star Compact automated system using Auto Histone ChIP-seq kit (Diagenode s.a., Belgium). A minimum of two IPs of 10^6 cells (2 x 10^6 in case of H3K27ac) per cell line was used. Replicates were pooled following RNase A and proteinase K treatments. DNA was purified with Qiagen DNA purification kit. DNA concentration was measured using Qubit apparatus (Life Technologies). Before proceeding with library preparation for sequencing, enrichment of the precipitated DNA was assessed by quantitative PCR. Of note, automatization of the procedure to reach the necessary throughput required by the project did not significantly modify the results. Paralleled chromatin IP of 10^7 cells performed manually using Dynabeads magnetic beads (Life Technologies) to collect chromatin-antibody complexes showed concordant results. 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
NA12891
Experimental Factor: population
HapMap CEU
Experimental Factor: immunoprecipitate
H3K4me3