GSM7051084: ARID1A_CnR_CSTantibody_cKO1; Mus musculus; OTHER
Aliquots of cryopreserved Arid1aWT and Arid1acKO  pachytene spermatocytes were quickly thawed in a water bath at room temperature (RT), pelleted at 600g for 5 minutes, and washed [wash buffer: 20mM HEPES(Na) pH7.5, 150 mM NaCl, 0.5 mM spermidine, EDTA-free protease inhibitor] three times. Cells were gently resuspended in wash buffer and bound to Concanavalin-A coated magnetic beads (20 µL beads/500,000 cells) by mixing on a rotator for 15 minutes. The bead slurry was separated using a magnet followed by permeabilization in 50 µL antibody buffer (wash buffer + 0.05% Digitonin + 2mM EDTA) per sample in 0.2 mL 8-strip PCR tubes. Primary antibody was added to the samples and incubated on a nutator at 4 oC overnight. Cells were then separated using a magnet, washed two times in 200 µL Dig-wash buffer (wash buffer + 0.05% Digitonin), and incubated with a secondary antibody prepared in 50 µL of Dig-wash buffer/sample for 30 minutes on a nutator at 4 oC. After antibody incubations, samples were washed twice in 200 µL Dig-wash buffer and then incubated on a nutator at 4 oC with 50 µL Dig-wash buffer containing 1000ng/mL Protein A/G Micrococcal Nuclease (pA/G-MNase) for 1 hr. Following this, samples were washed twice in 200 µL Dig-wash buffer, resuspended in 50 µL Dig-wash buffer, cooled down to 0 oC, and supplemented with CaCl2 at a final concentration of 2 mM to activate MNase. Samples were digested at 0 oC for 40 minutes. The resulting chromatin fragments were extracted from the bead slurry by incubating in 2X STOP buffer (340 mM NaCl, 20mM EDTA, 4mM EGTA, 0.05% Digitonin, 100 µg/mL RNase A) at 37 oC for 30 minutes. DNA was purified using ChIP DNA clean and concentrator kit (Zymo, D5205). Libraries were created using Kapa hyperprep kit (Roche catalog no.  KK8504) for Illumina sequencing.