GSM5970934: RAD21 WT siGLO, Rep1; Mus musculus; ChIP-Seq
mESCs were crosslinked in 1% formaldehyde for 5 minutes before quenching with 2.5M glycine. After chromatin extraction, 5% of HEK293T chromatin was added to the mESC samples to be later used as a spike-in normalization for the indicated experiments. Crosslinked cells were lysed using Lysis Buffer 1 and Lysis Buffer 2 before washing and resuspension in Shearing Buffer. Sonication of nuclei was performed on a Covaris E220 with the following settings: Duty Factor 5, PIP/W 140, and 200 cycles per burst for 12 minutes. Chromatin fragments of 200-1000 base pairs were generated. The antibodies were incubated with 30uL and 50uL Protein G Dynabeads for RAD21 and PDS5 IPs respectivly, for 6 hours prior to addition of chromatin. The chromatin in shearing buffer was spiked with NaCl and Triton X-100 to be in a ChIP buffer. Chromatin from 1 x 10^7 cells was added to antibody conjugated beads and incubated rotating overnight at 4C. Beads were then washed with ChIP Buffer 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. The next day, DNA was purified using a Zymo ChIP DNA Clean and Concentrate kit. Sequencing libraries were prepared using Kapa Hyper Prep Kit. Sequencing libraries were prepared using Kapa Hyper Prep Kit (Kapa Biosystems).