title: GSM8334250: WT_Rad21_2; Mus musculus; ChIP-Seq
library_construction_protocol: 20 million RAG-deficient v-Abl cells treated with STI-571 for 4 days were collected, 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). ChIP DNA was purified with SPRI AMPure XP beads. 20ul Purified DNA (from above), 25μl High-Fidelity 2X PCR Master Mix (NEB, M0541), 2.5 μl N5xx (Vazyme, TD202), 2.5 μl N7xx (Vazyme, TD202). PCR was set up as follows: 98 °C, 30 s; [98 °C, 10 s, 60 °C, 20 s, 72 °C, 20 s] × 10-14 cycles; 4 °C hold. The amplified libraries were purified by AmpureXP beads and were sequenced via paired end 150bp sequencing on a NovaSeq 6000 (Illumina) sequencer.