Instrument: Illumina HiSeq 4000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: mESCs were crosslinked in 1% formaldehyde for 2 minutes before quenching with 125 mM glycine. Crosslinked cells were lysed using Lysis Buffer 1 and Lysis Buffer 2 before resuspension in Sonication Buffer 1. Human chromatin (from HEK293T cells) was spiked in (to final 5%) prior to sonication for the indicated experiments. Sonication of nuclei was performed on a Covaris E220 with the following settings: Duty Factor 8, PIP/W 210, and 200 cycles per burst for 12 minutes. Chromatin fragments of 200-1,000 base pair size were generated. Antibody was incubated with 100uL Protein G Dynabeads for 6 hours. Unbound antibody was removed via washing, as detailed above, before incubation of antibody bound beads with chromatin overnight. Beads were then washed with Sonication Buffer 1 and Wash Buffers 1, 2, and 3. Chromatin was eluted and crosslinks were reversed overnight via incubation at 65C and addition of 5ul Proteinase K. Zymo ChIP DNA Clean and Concentrate kit (D5205) was used to purify DNA following Proteinase digestion. Sequencing libraries were prepared using a Hyper Prep kit (Kapa Biosciences KK8502).