GSM9863664: CEBPB, biol rep2; Homo sapiens; OTHER
We adapted the CUT&RUN protocol and all buffer recipes from Skene and colleagues (Skene et al., 2018) and performed the experiments in 0.2 mL tubes as we recently described (Ishak et al., 2025). Briefly, we washed and immobilized 200 000 K562 cells on 10 μL of activated concanavalin A–coated magnetic beads (Polysciences BioMag®Plus cat. no. 86057-3). We incubated cells in 0.2 mL antibody buffer containing 1: 100 diluted primary antibody (anti-C/EBP𝛽, Santa Cruz Biotechnology (Dallas, Texas, USA) cat. no. sc-7962, anti-ZNF143 Proteintech (Rosemont, IL, USA) cat. no. 16618-1-AP; RRID: AB_2218324, or anti-IgG , Abcam (UK) cat. no. ab37415; RRID: AB_2631996) for 2 h at 4 °C with rotation. Then, we washed the cells with 0.2 mL of digitonin wash buffer. Next, we incubated the cells in 0.2 mL of digitonin wash buffer containing recombinant pA-MNase (expressed and purified in house) diluted to a final concentration of ∼ 0.8 ng/μL for 1 h at 4 °C with rotation. We then discarded the buffer and replaced it with 150 μL of digitonin wash buffer. Using an ice-water bath, we cooled the tubes to 0 °C for 5 min and added CaCl2 to activate pA-MNase-mediated digestion for 30 min. After 30 min , we added 2x STOP buffer containing chelating agents and heterologous Saccharomyces cerevisiae spike-in DNA to stop the reaction. We incubated the cells at 37 °C for 10 min and centrifuged the cells at 16 000 × g for 5 min at 4 °C. We isolated the DNA from the supernatant containing soluble chromatin fragments via phenol chloroform extraction. We performed library preparation of 5 ng of CUT&RUN DNA using NEBNext Ultra II DNA Library Preparation Kit for Illumina ( NEB, cat. no. E7645L) as per manufacturer's protocol. We uniquely indexed each sample using NEBNext Multiplex Oligos for Illumina (Index Primers Set 1, cat. no. E7335L and Index Primers Set 2, cat. no. E7500L). We sequenced all libraries at Princess Margaret Genomics Centre (PMGC, Toronto, ON) on an Illumina NextSeq 500 High-Output flow cell using paired-end 2 × 150 bp read length configuration.