title: GSM8127390: Smarca5_2; Mus musculus; OTHER
library_construction_protocol: Sorted B cells were subjected to the CUT&RUN protocol Version 3 (Janssens et al. 2019). B cells were added to a Concanavalin A-coated bead slurry that had been pre-incubated with CaCl2 and MnCl2 to activate the beads. The cells and beads were rotated for 10 minutes at room temperature to ensure that all cells were coated onto the beads. The cells of each mouse were divided into two samples. Tubes were then placed into a magnetic stand to remove the liquid, and the supernatants were discarded. The cell-bead mixtures were resuspended in 100 µl of antibody buffer containing 0.05% digitonin for permeabilization. Next, 1 µl of monoclonal rabbit-anti mouse SNF2H antibody (Abcam) or a rabbit monoclonal isotype antibody (Cell Signaling Technologies) were added to the permeabilized samples, and the mixture was incubated overnight on a rotator. The following day, the beads were washed twice and then incubated for one hour in 150 µl of digitonin-containing wash buffer with 700 ng/ml of pA/G-MNase (kindly provided by the lab of Steven Henikoff). After incubation, the beads were washed twice and resuspended in 100 µl of digitonin-containing wash buffer. The beads were then chilled to 0°C in a metal block resting in ice. Finally, 2 µl of 100 mM CaCl2 was added to activate the MNase activity, resulting in a final concentration of 2 mM. The reaction was stopped after 30 minutes. The bead-cell mixture was incubated for at least 30 minutes at 37°C to release restricted DNA fragments. Then, the tubes were placed into a magnetic stand, and the supernatants containing DNA fragments of interest were collected. DNA fragments were used directly for library preparation, by performing joint end repair and poly A tailing, Truseq adapter ligation and PCR enrichment with the KAPA HiFi master mix (Roche) for 10-14 cycles. CUT and RUN libraries were then analyzed with a Nova-Seq 6000 sequencer (Illumina) using paired end sequencing.