title: GSM9118509: Jurkat_3B6_TripleKO_H3K27ac; Homo sapiens; OTHER
library_construction_protocol: For nuclei preparation, cells were permeabilized for 10 min at 4°C in NE1 buffer (20 mM Hepes-KOH pH 7.9, 10 mM KCL, 0.5 mM Spermidine, 0.1% Triton-X100, 20 % Glycerol, protease inhibitor cocktail). Then nuclei were washed with 20 mM Hepes pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, protease inhibitor cocktail). The protocol was adapted from Carter et al (PMID: 31431618). The protein A (pA) fused to Tn5 Transposase (pA-Tn5) was first loaded with DNA duplexes (pMENTS oligonucleotide with either I'5 or I7' adapter oligonucleotides) and then incubated with antibodies targeting H3K27Ac, H3K4Me1 or H3K4Me3. Next, pA-Tn5-Antibody complexes were incubated with nuclei at RT for 1 h. Tagmentation was initiated by adding MgCl2 (incubation 1 h, 37°C). The reaction was stopped by adding EDTA, SDS and Proteinase K (55°C for 1 h) and DNA was purified using Zymo-Spin IC Columns. For library preparation, DNA (250 ng) were amplified with i'7 and i'5 primers and  NEBNext High-Fidelity 2X PCR Master Mix; the following PCR programwas used: 72°C for 5 min; 98°C for 45 sec; 22 cycles of [98°C for 15 sec, 63°C for 10 sec]; 72°C for 1 min; hold at 8°C. PCR products (CUT&Tag libraries) were separated by gel electrophoresis and DNA fragments between ~250-1000 bp were excised and purified.