title: GSM8377795: MCF7_ChIP_RNAPol2_SNDX_200nM_24h_REP1_240307_CAGATC_S7; Homo sapiens; ChIP-Seq
library_construction_protocol: The cells were collected and washed twice in ice-cold PBS followed by crosslinking in 1% formaldehide (methanol free) for 10 minutes. Excess of formaldehyde was quenched by addition of Tris pH 8.0 to 100nM final and Glycine 125 mM final. Chromatin was sheared using Covaris E220 for 30 min with power 140; duty 5; bursts 200/sec. We then used a 5ul aliquot of sherared chromatin to decrosslink in order to test completeness of shearing. Chromatin passed QC if fragments between 100-700bp were >90%. For immunoprecipitation we used chromatin from 5-20x10^6 cells, antibody and Protein-A/G magnetic beads. Following roatation at 4 degrees overnight, beads were wased in Mixed Micelle Buffer, buffer 500, LiCl/detergent solution and TE buffers. DNA was eluted from the beads using 100mM NaHCO3 100mM NaCl 1% SDS and purified using AmpureXP beads. 1-10 ng of DNA was used for the library preparation using the ThruPLEX DNA-Seq Kit (Takara) ChIP-sequencing was done using the Illumina Next Gen Sequencing NextSeq platform (Illumina, San Diego, CA) to obrain 25-30 million 37bp, paired-end reads.