MCF-7/shCtrl_ER_rep1
Bead-antibody complexes were prepared by mixing ChIP-Grade Protein G Mag-netic Beads (Cell Signaling Technology) with ER antibody (Cell Signaling Technology #8644) at 0.079 mg/mL concentration and rotating at 4C overnight. Bead-antibody complexes were washed and resuspended with cold blocking buffer (0.5% BSA in PBS), and then bead suspension was added to sonicated chromatin and IP was performed by rotatioin at 4C overnight. Bead-antibody-chromatin mix was washed 3x with blocking buffer. Immunoprecipitates were tagmented using the ChIPmentation procedure (Schmidl et al., 2015). DNA was eluted and crosslinks were reversed at 65C overnight. DNA was treated with RNase A and Proteinase K, then purified using Quick-DNA Microprep Kit (Zymo Research). DNA was amplified with 11 PCR cycles using custom barcoded primers (Nextera), quality control-checked on a Frag-ment Analyzer (Agilent), quantified by Qubit, and pooled for sequencing on a NextSeq2000 (Illumina). Libraries were sequenced using 50-bp paired-end reads targeting 50M reads per sample. Sequencing reads were processed with the ENCODE Histone ChIP-seq pipeline (v2.2.1) (github.com/ENCODE-DCC) and aligned to the human genome (hg38) using Bowtie2 (v2.5.1) with minimum read length set at 2 (Langmead and Salzberg, 2012). Unmapped/low quality reads were filtered using samtools (v1.16.1) and duplicated reads were removed using Picard (v2.27.5).