title: GSM5505498: CHG213; Homo sapiens; ChIP-Seq
library_construction_protocol: For ChIP-seq, 1 million fresh harvested cells were fixed in 1% formaldehyde/medium buffer for 10 minutes (min) at room temperature. Fixation was stopped by addition of glycine to a final concentration of 125 mM. Fixed cells were washed 3 times with TBSE buffer, and centrifuged (5,000 r.p.m., 5 min). Pelleted cells and pulverized tissues were lysed in 100 µl 1% SDS lysis buffer and sonicated to 300-500bp using a Bioruptor (Diagenode). ChIP was performed using the following antibodies: H3K4me3 (07-473, Millipore); H3K4me1 (ab8895, Abcam); H3K27ac (ab4729, Abcam). For MeDIP-seq, DNA was sonicated using COVARIS S2 and peak fragment distribution between 100-500bp was verified on an Agilent Bioanalyzer (Agilent Technologies) using the DNA1000 chip.  Fragmented DNA was end-repaired, dA-tailed and adapter ligated using NEBNext® DNA Library Prep Master Mix Set for Illumina (E6040). Samples were then spiked with control DNAs that were unmethylated, methylated and hydroxymethylated (Diagenode C02040010) as a quality control measure.  For each sample, input DNA that was not exposed to the primary antibody was included.  Adapter-ligated DNA was subjected to immunoprecipitation with a primary monoclonal antibody against 5-methyl cytosine (Diagenode C15200081) For ChIP-seq, 30ng of amplified DNA was used for each sequencing library preparation (New England Biolabs). 8 libraries were multiplexed (New England Biolabs) and sequenced on 2 lanes of a Hiseq2500 sequencer (Illumina) to an average depth of 20-30 million reads per library. For MeDIP-seq, Immunoprecipitated samples were amplified using Phusion® High-Fidelity DNA Polymerase (M0530) and NEBNext® Multiplex Oligos for Illumina® (E7335) for 10 cycles. Amplified libraries were run on the Agilent Bioanalyzer using the High sensitivity DNA kit prior to Illumina sequencing using a single-end 100 base pair configuration.