GSM8741992: Kat6a WT Kat6b WT, H3K23ac, rep 3; Mus musculus; OTHER
Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing was performed on 50,000 mouse embryonic fibroblasts (MEFs) combined with 50,000 Drosophila melanogaster S2 cells, as described in Kaya-Okur et al., 2019, with minor modifications as described in. All buffers were prepared fresh with complete EDTA-free protease inhibitors (Roche, 11873580001) and 0.5 mM sodium butyrate (Sigma, B5887). Per sample, 10 µl concanavalin-A beads (Bangs Laboratories, BP531) were washed twice in 10 volumes binding buffer (20 mM HEPES pH 7.5 (Sigma, 83264), 10 mM KCl (Sigma, 60142), 1 mM CaCl2 (Sigma, 21115), and 1 mM MnCl2 (Sigma, M1787) and resuspended in 10 µl binding buffer. Combined MEFs and S2 D. melanogaster cells were washed twice in 1 ml wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl (Sigma, 71386), 0.5 mM spermidine (Sigma, S0266) and resuspended in 90 µl wash buffer. Concanavalin-A beads and samples were combined and incubated (RT, 10 min), immobilised using a magnetic rack (ThermoFisher, MR02) and beads resuspended in 100 µl ice-cold antibody buffer (wash buffer supplemented with 0.05 % (w/v) digitonin (Merck, 300410), 2 mM EDTA (Invitrogen, 15575020), and 0.1 % (w/v) BSA (Sigma, A8577) containing 1:100 H3K23ac (Millipore, 07-355) primary antibody. Samples were incubated overnight at 4oC. The following morning, beads were resuspended in 100 µl wash buffer supplemented with 0.05% (w/v) digitonin containing 1:100 secondary antibody (Guinea pig anti-rabbit IgG, Antibodies Online, ABIN101961), incubated (RT, 1 hr), washed thrice and resuspended in 100 µl ice-cold dig-300 buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM spermidine and 0.01 % (w/v) digitonin), supplemented with 1 2.5 µl pAG-Tn5 (EpiCypher, 15-1017). Samples were incubated (RT, 1 hr), washed thrice, resuspended in 100 µl tagmentation buffer (wash buffer supplemented with 0.01 mM MgCl2 (Sigma, 63069) and incubated at 37°C for 1 hr. Tagmentation was stopped using 3.34 µl 0.5 M EDTA, 1 µl 10% (w/v) SDS (Sigma, 71736), and 0.83 µl 20 mg/ml thermolabile proteinase K (NEB, P8111S), incubating at 37°C for 1 hr and 800 rpm, followed by heat inactivation at 55°C for 10 min. DNA was extracted using Ampure XP beads (Beckman, A63880), eluting in 25 µl 10 mM Tris-HCl pH=8.0 (Invitrogen, 15568025), 1 mM EDTA, and 25 µg/ml RNAse A (ThermoFisher, EN0531) at 37°C for 10 min. 10 µl sample DNA elutes were used to generate sequencing libraries, which were amplified using PCR for 13 cycles. PCR products were cleaned up using 30 µl Ampure XP beads and eluted in 25 µl 10 mM Tris-HCl pH 8.0. Cleaned libraries were analysed using High Sensitivity D1000 gels (Agilent, 5067- 5584) on an Agilent 2200 tape station.