title: GSM8718563: HME1_MSL3ko_CUT&Tag_Rep1; Homo sapiens; OTHER
library_name: GSM8718563
library_construction_protocol: We performed two biological replicates of CUT&Tag on HME1 cells. Cells were pelleted by centrifugation for 3 min at 600 x g at room temperature and resuspended in 500 μL of ice-cold NE1 buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 0.5 mM spermidine, 1% Triton-X-100, and 20 % glycerol and cOmplete EDTA free protease inhibitor tablet) and was let to sit for 10 min on ice followed by nuclei preparation. Nuclei were pelleted by centrifugation for 4 min 1300×g at 4 °C and resuspended in 500 μl of wash buffer and by placing the tubes on a magnet stand to clear and withdraw the liquid, then resuspended in 1.0 ml wash buffer and held on ice until beads are ready. For each tube, 10 μl of BioMag Plus Concanavalin-A-conjugated magnetic beads (ConA beads, Polysciences, Inc) were resuspended in the binding buffer and was kept on nutator for 10 minutes. After a pulse spin to remove liquid from the cap, tubes were placed on a magnet stand to clear, the liquid was withdrawn, and we next added primary antibody (1ul per sample) resuspended in 800 μl of antibody buffer for targeting H4K16ac (Abcam, ab109463) or IgG (normal rabbit IgG, Santa Cruz Cat no sc-2027) and incubated overnight at 4 °C on a nutator. Next day, secondary antibodies (guinea pig α-rabbit antibody, Antibodies online cat. no. ABIN101961) were added 1:100 in Dig-wash buffer (5% digitonin in wash buffer) and squirt in 100 μl per sample while gently vortexing to allow the solution to dislodge the beads from the sides and incubated for 60 min on a nutator. We got rid of any unbound antibodies by 3X wash with 1 mL of dig-wash buffer. The complex was then incubated with 100 μl of (1:250 diluted) protein-A-Tn5 loaded with adapters (prepared in the lab) in Dig-300 buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM spermidine with Roche cOmplete EDTA-free protease inhibitor) and placed on a nutator for 1 h followed by three washes with 1 ml of Dig-300 buffer to remove unbound pA-Tn5. Tagmentation was carried out by adding 300 μl Tagmentation buffer (Dig-300 buffer + 5 mM MgCl2) gently while mild vortexing and incubating for 1hr at 37 °C. Tagmentation was stopped by adding 10 µL 0.5 M EDTA, 3 µL 10% SDS and 2.5 µL 20 mg/mL Proteinase K to each sample. Samples were mixed by full-speed vortexing for ~2 s and incubated for 1 h at 55 °C to digest proteins. DNA was extracted by phenol: chloroform protocol using phase-lock tubes (Quanta Bio) followed by ethanol precipitation. Libraries were prepared using NEBNext HiFi 2x PCR Master mix (Cat number M0541S) with 72 °C gap filling step followed by 13 cycles of PCR with 10-s combined annealing and extension for enrichment of short DNA fragments. Libraries were sequenced in Novaseq 6000 (Novogene) with 150bp paired-end reads at Novogene sequencing service.