title: GSM2897187: ChIP-seq_HAP1_WT_Input_8x_r1; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIPmentation was carried out as described (Schmidl et al, Nature Methods 2015). Cells were washed once with PBS and fixed with 1% paraformaldehyde in up to 1ml PBS for 10 min at room temperature. Glycine was added to stop the reaction. Cells were collected at 500 g for 10min at 4 C, washed twice with cold PBS supplemented with 1 mM phenylmethyl sulfonyl fluoride (PMSF). The pellet was lysed in sonication buffer (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 0.25% SDS, 1uL protease inhibitors (Sigma) and 1 mM PMSF) and sonicated with a Covaris S220 sonicator to a range of 200-700 bp. Lysates were centrifuged at full speed for 5min at 4 C, and the supernatant transferred to a new tube. The lysate was then brought to RIPA buffer conditions (final concentration: 10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 140mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, 1uL protease inhibitors (Sigma) and 1 mMPMSF) to a volume of 200 ml per IP. For each immunoprecipitation, 10 ml magnetic Protein A (Life Technologies) were washed twice and resuspended in PBS supplemented with 0.1% BSA. The antibody was added and bound to the beads by rotating 2 h at 4 C. Used antibodies were H3K4me1 (0.5 mg per IP, Diagenode pAb-194-050), H3K27ac (1 mg per IP, Diagenode pAB-196-050) and H3K27me3 (1 mg per IP, Millipore 07-499). For control libraries, an IP with 2.5 mg of a nonspecific IgG rabbit antibody was used. Blocked antibody-conjugated beads were then placed on a magnet, supernatant was removed and the sonicated lysate was added to the beads followed by incubation for 3-4 h at 4 C on a rotator. Beads were washed subsequently with RIPA (twice), RIPA-500 (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 500mM NaCl, 1% Triton X-100, 0.1% SDS and 0.1% DOC) (twice) and RIPA-LiCl (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 250mM LiCl, 1% Triton X-100, 0.5% DOC and 0.5% NP40) (twice). Beads were washed once with cold Tris-Cl pH 8.0, to remove detergent, salts and EDTA. Beads were washed once more with cold Tris-Cl pH 8.0. DNA was purified with SPRI AMPure XP beads (sample-to-beads ratio 1:2) or Qiagen MinElute columns. One microlitre of each library was amplified in a 10-ml qPCR reaction containing 0.15 mM primers, 1uL SYBR Green and 5 ml Kapa HiFi HotStart ReadyMix (Kapa Biosystems), to estimate the optimum number of enrichment cycles with the following programme: 72 oC for 5min, 98 oC for 30 s, 24 cycles of 98 oC for 10 s, 63 oC for 30 s and 72 oC for 30 s, and a final elongation at 72 oC for 1min. Kapa HiFi HotStart ReadyMix was incubated at 98 oC for 45 s before preparation of all PCR reactions (qPCR and final enrichment PCR). Final enrichment of the libraries was performed in a 50-ml reaction using 0.75 mM primers and 25 ml Kapa HiFi HotStart ReadyMix. Libraries were amplified for N+1 cycles, where N is equal to the rounded-up Cq value determined in the qPCR reaction. Enriched libraries were purified using SPRI AMPure XP beads at a beads-to-sample ratio of 1:1, followed by a size selection using AMPure XP beads to recover libraries with a fragment length of 200-400 bp. Library preparation was performed using custom Nextera primers as described for ATAC-seq. The libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq 4000 platform and the 50-bp single-end configuration.
attributes.tag: GEO Accession
attributes.value: GSM2897187