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 (single-end sequencing, read length 36 bp). Pilot2 samples (excluding H3K27ac) were sequenced one sample per lane. Pilot1 samples (and pilot2 samples for H3K27ac) were multiplexed and sequenced either three (POLR2B, H3K27me3, and pilot2 H3K27ac) or four samples per lane (all other samples). H3K4me1 pilot1 libraries were sequenced twice in order to increase the coverage.
[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.
nucleic acid sequencing protocol
Sequencing was performed on the Illumina Genome Analyzer IIx platform using TruSeq PE Cluster Kit v3 (cluster generation) and TruSeq SBS Kit v5 (single-end sequencing, read length 36 bp). Samples were sequenced one sample per lane.
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.
nucleic acid library construction protocol
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).
[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.
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.
normalization data transformation protocol
Raw sequence reads were mapped against the standard hg19 build of the human reference genome using BWA 0.5.9 (http://bio-bwa.sourceforge.net) with default parameters for single-end reads. Uniquely mapping reads with a mapping quality >=10 were kept for further analyses.