title: GSM1434978: PD32 H4K20me3 ChIP-seq (Ab1); Homo sapiens; ChIP-Seq
library_construction_protocol: Cross-linked cells were pre-extracted with (1:1) modified nuclear lysis buffer (mNLB):IP dilution buffer (IPDB) [35 mM Tris-HCl pH 8.0, 75 mM NaCl, 5.5 mM EDTA pH 8.0, 3 mM EGTA pH 8.0, 0.5% SDS, 0.5% Triton X-100] supplemented with 10 μg/ml aprotinin, 5 μg/ml leupeptin, 50 μg/ml PMSF and sonicated at a density of 2 x 107 cells per 1 ml cold (1:1) mNLB:IPDB plus inhibitors. Sonicated chromatin solutions were cleared by centrifugation, diluted with IPDB [20 mM Tris-HCl pH 8.1, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.01% SDS] to a final mNLB:IPDB ratio of 1:10, transferred to microcentrifuge tubes containing antibodies (from Millipore (04-079) and Cell Signaling Technology (5737)) pre-bound to Dynabeads M-280 Sheep anti-Rabbit IgG magnetic beads (Life Technologies), and incubated overnight at 4° C with rotation. The ChIP reactions were washed twice with IPDB, once with High Salt Wash Buffer [20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X- 100], once with LiCl Wash Buffer [10 mM Tris-HCl pH 8.1, 250 mM LiCl, 1 mM EDTA, 1% NP-40, 1% deoxycholic acid] and twice with 1X TE. Beads were aspirated to dryness, resuspended in 500 μl IP Elution Buffer [50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM EDTA pH 8.0, 1% SDS] and 0.5 μl 100 mg/ml RNase A and incubated at 65° C for 4-6 hours. To each tube, 6 μl 20 mg/ml proteinase K was added and the tubes were incubated at 45° C for 12 hours. ChIP DNA was purified by phenol/chloroform extraction with ethanol precipitation, resuspended with 20 μl nuclease-free dH2O and quantified using the Qubit dsDNA HS Assay Kit and a Qubit fluorometer (Life Technologies). Libraries were prepared according to NEB's instructions accompanying the NEBNext® ChIP-Seq Library Prep Master Mix Set for Illumina®(E6240S). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (3' to 5' exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were isolated by agarose gel excision and PCR-amplified using Illumina primers. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer IIx following the manufacturer's protocols.