title: GSM9010156: Input, YFP-BAF S2 cells, Replicate2; Drosophila melanogaster; ChIP-Seq
library_construction_protocol: S2 cells (14 × 10⁶) were crosslinked with 1.8% formaldehyde for 10 minutes at room temperature and chromatin was prepared as described in {Bujosa, 2024 #65}. Chromatin samples were sonicated using a Bioruptor Pico (Diagenode) to obtain fragments between 200–500 bp and, after sonication, lysates were adjusted to 1% Triton X-100, 0.1% sodium deoxycholate (DOC), 140mM NaCl, incubated for 10 minutes on a rotating wheel at 4°C, and chromatin was recovered by centrifugation. For each experiment, 400 μl of chromatin were used for IP, and 40 μl were retained as input. Immunoprecipitation (IP) was carried out in RIPA buffer (140mM NaCl, 10mM Tris–HCl, pH 8.0, 1mM EDTA, 1% Triton X-100, 0.01% SDS, 0.1% DOC). For endogenous BAF, lysates were pre-cleared with 30 μl of 50% (v/v) protein A–Sepharose CL-4B beads (GE Healthcare, 17-0780-01) pre-blocked with RIPA + 1% BSA and, after pre-clearing, 10 μl of aBAF antibodies were added, and samples were incubated overnight at 4°C with rotation. Then, IPs were performed by adding 40 μl of 50% (v/v) protein A–Sepharose CL-4B beads (pre-blocked with RIPA + 1% BSA) and incubating for 3 hours at 4°C on a rotating wheel. For N-BAF, C-BAF and N-BAFT4A/S5A samples, 40 μl of GFP-Trap Magnetic Agarose beads (Chromotek) were added, and samples were incubated for 3 hours at 4°C with rotation. After IP, beads from all conditions were washed five times for 5 minutes with 1 ml of RIPA buffer, once with high-salt buffer (250mM LiCl, 10mM Tris–HCl, pH 8.0, 1mM EDTA, 0.5% NP-40, 0.5% DOC), and twice with TE buffer for 5 minutes. Beads were resuspended in 40 μl of TE and RNase A (DNase-free) was added to a final concentration of 0.25 μg/ml to both IP and input samples, and samples were incubated for 30 minutes at 37°C. To purify DNA, samples were adjusted to 1% SDS, 100mM NaHCO₃, 0.2 mg/ml Proteinase K, and incubated overnight at 65°C. DNA was purified using phenol-chloroform extraction. Libraries were generated using NEBNext Ultra II DNA Library Prep Kit (Illumina) and sequenced by 2x150 paired-end reads on a NovaSeq S4 (Illumina) at the CNAG (Barcelona).