GSM9629001: IgG_ChIPseq_WT_rep1; Mus musculus; ChIP-Seq
About 10 million CH12F3 cells were fixed with 1% formaldehyde for 10 min at room temperature, and quenched with glycine at a final concentration of 125 mM. The pellet was sonicated with a Covaris M220 sonicator for 5-8 min in sonication buffer until the size of most fragments was in the range of 200–700 bp. 25ug sonicated chromatin was then brought to RIPA-LS buffer conditions. The antibody was added by rotating overnight at 4 °C. For each immunoprecipitation, 25 μl magnetic Protein A (Invitrogen, 10002D) were washed twice and resuspended in 500ul PBS supplemented with 5mg/ml BSA and incubate 4°C overnight to block beads. The blocked beads were added and bound to antibody by rotating 2 h at 4 °C. Used antibodies were Rad21 (0.2 μg per immunoprecipitation, abcam, ab992). Beads were washed subsequently with RIPA-LS (twice), RIPA-HS (twice) , RIPA-LiCl (twice) and 10 mM Tris pH 8.0 (once) to remove detergent, salts and EDTA. Beads were resuspended in 25 μl of the tagmentation reaction mix containing 1 μl Tagment DNA Enzyme and incubated at 37 °C for 5-10 min in a thermocycler. The beads were washed with RIPA-LS (twice) and once with TE. And then de-crosslinked with Proteinase K (Roche, #03115852001). Tagmented DNA was released from beads and purified with Zymo DNA Clean&Concentrator-5 kit. ChIP-seq libraries were constructed using NEBNext High-Fidelity PCR Master Mix (New England Biolabs, M0541) and illumina index primers. The final ChIP-seq libraries were size-selected by SPRIselect beads (Beckman Coulter, B23318) and sequencing via paired-end 150bp sequencing on Novaseq 6000 (Illumina).