Yy1 antibody

The ChIP protocol was conducted as described by Schmidt et al.58 (link) with few modifications. In summary, following fixation, the tumour tissue undergoes chromatin extraction and sonication using the Bioruptor Pico sonication device (Diagenode; B01060001) using 20 cycles (30s on and 30s off) at maximum intensity. Purified chromatin was then separated for 1. Immunoprecipitation using 4ug of H3k27ac antibodies (Abcam; ab4729) per ChIP experiment or using 4ug of YY1 antibodies (Santa Cruz; sc-281 X). ChIP-seq experiment for YY1 were performed in biological duplicates. Cells were stimulated with estrogen for 45 minutes, upon which maximum ERα-binding to chromatin occurs. Biological replicates showed very high correlation (R²=0.98), thus only consensus loci were kept for further analyses. 2. Non-immunoprecipitated chromatin, used as Input control and 3. Assessment of sonication efficiencies using a 1% agarose gel. Before construction of ChIP-seq libraries (NEB Ultra II kit, see supplementary methods), enrichment of the immunoprecipitated sample was ascertained using positive and negative controls for ChIP-qPCR. Library preparation was performed using 10 – 50 ng of immunoprecipitated and Input samples. Before sequencing, libraries were again re-tested to confirm enrichment using positive and negative controls.

Naïve splenic B cells were cross-linked with 1% formaldehyde for 10 min at room temperature. After cell lysis, chromatin was sheared by using a Covaris S220 for 20 min with 200 cycle/burst, 10 duty factor, and 140 peak power. Note that 100 μg of sheared chromatin was used per immunoprecipitation, and 1% was reserved as input control. Sheared chromatin was incubated overnight in 4°C with preblocked Dynabeads Protein G (Invitrogen) coated with YY1 antibody (#414; Santa Cruz Biotechnology). The following day, beads were washed and DNA was purified by using the QIAquick PCR Purification Kit. Note that 6 ng from each sample was used to construct ChIP-Seq libraries with a unique index using the TruSeq ChIP Library Preparation Kit (Illumina, San Diego, CA) according to the manufacturer’s instructions. ChIP-Seq reads were aligned to the mouse reference genome (mm10) using Bowtie 1.1.2. Duplicated reads were removed by Samtools. Significant YY1 peaks were called using MACS (v2.1.0), with a FDR ≤ 5%. Annotation of Chip-Seq data was performed by Homer. Bedtools were used to process the bed files and overlapping peaks were defined as peaks sharing more than 10% of the length of the shorter peak. ChIP-seq data are available in the Gene Expression Omnibus (GEO) database under the accession number GSE104097.