GSM6043386: H9, H3K4me3 Rep1; Homo sapiens; OTHER
CUT&Tag was performed according to the protocol of Kaya-Okur et al (1) with modifications to tissue processing as described below. ~100,000 cells were pelleted by centrifugation for 3 min at 600 x g at room temperature and resuspended in 500μl 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. Nuclei were pelleted by centrifugation for 4 min at 1300 x g at 4°C, resuspended in 500μl of wash buffer and held on ice until beads were ready. Required amount of BioMag Plus Concanavalin-A-conjugated magnetic beads (ConA beads, Polysciences, Inc) were transferred into binding buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 1mM CaCl2 and 1mM MnCl2), washed once in the same buffer, each time placing them on a magnetic rack to allow the beads to separate from the buffer and resuspended in binding buffer. 10 μl of beads was then added to each tube containing cells and rotated on an end-to-end rotator for 10 minutes. After a quick spin to remove liquid from the cap, tubes were placed on a magnet stand to clear, the liquid was withdrawn and 800μl of antibody buffer containing 1μg of primary antibodies: normal rabbit IgG (Santa Cruz Cat no sc-2027), H3K27ac (Abcam, ab4729), H4K16ac (Abcam, ab109463), H3K122ac (Abcam, ab33309), H3K4me1(Abcam, ab8895), H3K36me3 (Abcam, ab9050)) H3K4me3 (Millipore, 07-473),  H3K27me3 (Abcam, ab192985) and H3K9me3 (Abcam, ab176916)) was added and incubated at 4ºC overnight in a nutator. Secondary antibodies (guinea pig α-rabbit antibody, Antibodies online, ABIN101961) were added 1:100 in Dig-wash buffer (5% digitonin in wash buffer) and 100 µL was squirted in per sample while gently vortexing to allow the solution to dislodge the beads from the sides and incubated for 60 min on a nutator. Unbound antibodies were washed in 1 ml of Dig-wash buffer three times. 100 μl of (1:250 diluted) protein-A-Tn5 loaded with adapters in Dig-300 buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM spermidine with Roche cOmplete EDTA free protease inhibitor) was added to the samples, placed on nutator for 1 hour and washed three times in 1 ml of Dig-300 buffer to remove unbound pA-Tn5. 300 µL Tagmentation buffer (Dig-300 buffer + 5 mM MgCl2) was added while gentle vortexing and samples were incubated at 37ºC for 1 hr on an incubator. Tagmentation was stopped by adding 10 µL 0.5M 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 seconds and incubated for 1 hr at 55ºC to digest proteins. DNA was purified by phenol: chloroform extraction using phase-lock tubes (Quanta Bio) followed by ethanol precipitation. Libraries were prepared using NEBNext HiFi 2x PCR Master mix (M0541S) with a 72ºC gap-filling step followed by 13 cycles of PCR with 10-second combined annealing and extension for the enrichment of short DNA fragments. Libraries were sequenced in Novaseq 6000 (Novogene) with 150bp paired-end reads.