title: GSM6260175: MDA-MB-231 sgNT1, H3K4me3, biological replicate1; Homo sapiens; ChIP-Seq
library_construction_protocol: Around 2 x 107 cells and two biological repeats per genotype were used for ChIP-seq experiment. Crosslinking was performed with 1% formaldehyde (Sigma, F1635) at 37°C for 10 min. A final concentration of 125 mM glycine was added for 5 minutes at room temperature to quench formaldehyde crosslinking. Cells were washed with cold PBS and harvested on ice. Cells were lysed with 1ml of SDS lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl pH8) in the presence of protease inhibitor (Roche, cat#11836170001) and phosphatase inhibitor (Thermo, cat#78427). Cells were sonicated using the Branson Sonifier 250 (Branson Ultrasonics 101063588) with a 20% amplitude setting for 5 min 30 s with 10 s of on and off intervals. After sonication, 1 ml of ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH8, 167 mM NaCl) with proteinase and phosphatase inhibitor was added to prevent SDS precipitation. After centrifugation at 14,000 rpm for 10 min at 4°C, 2 ml of supernatant were transferred to a 15 ml falcon tube and diluted with 3 ml of ChIP dilution buffer containing proteinase and phosphatase inhibitor.  50 μl of Dynabeads (Thermo Fisher Scientific 10009D), which were previously blocked with 1% BSA, were added to samples and incubated at 4°C with rotation for 1 hour. After pre-clearing, Dynabeads beads were removed. The supernatant was transferred to new 15 ml falcon tubes and 100 μl out of 5 ml volume was collected as 2% input. Antibodies were added to the pre-cleared samples for overnight incubation at 4°C with rotation. 200 μl of Dynabeads that were previously blocked with 1% BSA, were added to the sample and incubated for 6 hours at 4°C with rotation. After incubation, beads were collected and washed at 4°C with rotation following the procedure: 5 min with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH8, 150 mM NaCl), twice 5 min with high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH8, 500 mM NaCl) and twice 5 min with TE buffer (10 mM Tris-HCl pH8, 1 mM EDTA). After washing, beads were resuspended in 250 μl of elution buffer (1% SDS, 0.1 M NaHCO3) and incubated at 60°C for 30 min using a thermomixer (850 rpm). The supernatant was transferred to new tubes and 5 M NaCl was added for overnight de-crosslinking at 65°C. 10 μl of 0.5 M EDTA, 20 μl of 1 M Tris-HCl (pH 6.5) and 1 μl of proteinase K (20 mg/ml, NEB, P8107S) were added and incubated at 45°C for 1 hour for de-crosslinking. ChIP-DNA was isolated by using the QIAquick PCR purification kit (QIAGEN, 28104). Libraries were prepared using the NEBNext® Ultra II DNA Library Prep Kit (NEB, E7103S) and NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1; NEB, E7335S). Samples were pooled and submitted to MSKCC Integrated Genomics Operation core for sequencing. Libraries were run over one lane of a HiSeq 4000 in a 50bp/50bp paired end run, using the HiSeq 3000/4000 SBS Kit (Illumina). 