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. [PU.1, MYC, H3K4me1]: Cells were lysed in nuclei extraction buffer (50 mM HEPES-NaOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8.0, 10% glycerol, 0.5% NP-40, 0.25% TritonX-100) supplemented with a protease inhibitor tablet (Roche) and phosphatase inhibitors (5 mM NaF, 1 mM _-glycerol phosphate and 1 mM sodium orthovanadate) for 10 min at 4 degrees C on a shaker. The isolated nuclei were then washed using washing buffer (200mM NaCl, 1mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0, 10 mM Tris-HCl pH 8.0) supplemented with protease and phosphatase inhibitors at RT for 10 min. Washed nuclei were resuspended in sonication buffer (1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0, 10 mM Tris-HCl pH 8.0 and 1% TritonX-100) containing protease and phosphatase inhibitors and the chromatin was fragmented using a Bioruptor sonicator (Diagenode) for 80 min using high amplitude and 30s ON and 30s OFF cycles to obtain 200-500 bp-sized fragments. The fragmented chromatin was then centrifuged at 17,000xg for 5 min and clear supernatant was diluted with ChIP dilution buffer (1 mM EDTA pH 8.0, 10 mM Tris-HCl pH 8.0 and 1% TritonX-100 containing protease and phosphatase inhibitors) to get chromatin equivalent to 10 x 10^6 cells for each IP. All IPs were performed in duplicates. BSA and ssDNA (Salmon Sperm DNA)-preblocked protein-A sepharose (80 ul/IP) beads were added to the samples and incubated for 2h to remove non-specifically binding chromatin. To the supernatant, 5 ug/IP rabbit polyclonal anti-Myc antibody was added to immunoprecipitate the chromatin complex at 4 degrees C overnight. After incubation, 50 ul blocked protein-A sepharose beads were added to each sample and incubated for 90 min at 4 degrees C to pull down the respective antibody-chromatin complexes. The beads were then washed four times with low salt wash buffer (20 mM Tris-Cl pH 8.0, 150 mM NaCl, 2 mM EDTA pH 8.0, 0.1% SDS, 1% TritonX-100) followed by two washes with high salt wash buffer (20 mM Tris-Cl pH 8.0, 500 mM NaCl, 2 mM EDTA pH 8.0, 0.1% SDS, 1% TritonX-100), lithium chloride wash buffer (10 mM Tris-Cl pH 8.0, 0.25 M LiCl, 1 mM EDTA pH 8.0, 1% NP-40, 1% sodium deoxycholate) and Tris-EDTA (TE) buffer (10 mM Tris-Cl pH 8.0, 1 mM EDTA pH 8.0). The c-Myc-bound chromatin complexes were eluted from beads for 30 min using 200 ul of elution buffer (100 mM sodium bicarbonate and 1% SDS in milliQ water). The chromatin was then reverse-crosslinked at 65 degrees C overnight after adding 8 ul of 5 M NaCl. The DNA was purified from the reverse-crosslinked chromatin by proteinase-K and RNase digestion followed by purification using Qiagen DNA purification columns. The purified DNA was eluted in 30ul of Qiagen elution buffer. PU.1 and H3K4me1 ChIPs were performed with slight modifications in the protocol described above. We used 1% SDS instead of TritonX-100 in sonication buffer to increase the stringency of chromatin pull-down by the respective antibodies and the sonication was performed for 60 min instead of 80 min. 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
NA19238
Experimental Factor: population
HapMap YRI
Experimental Factor: immunoprecipitate
PU.1