GSM8711911: siBAZ2A Replicate 4; Homo sapiens; OTHER
Briefly, 5 x 105 (G4 CUT&Tag) or 1 x 106 (HA CUT&Tag) cells were harvested per condition, washed with PBS and resuspended in NP40-digitonin buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.05% digitonin, 0.01% NP40 and Roche cOmplete Protease Inhibitor EDTA-free). The cells were then centrifuged at 600 x g for 3 min, resuspended in 200 mL antibody buffer (NP40-digitonin buffer with 1% BSA, for the BG4 step only, 2 mM EDTA is added) containing 2 mg of BG4 (purified from E. coli), anti-H3K4me3 Ab (G4 CUT&Tag: EDM Millipore, MC315; HA CUT&Tag: EpiCypher, 13-0060) or anti-HA Ab (EpiCypher, 13-2010) and 35 ng Drosophila Spike-in Chromatin (Active Motif, 53083) and incubated overnight at 4°C. Nuclei were washed once with NP40-digitonin buffer followed by centrifugation at 600 x g for 4 min, whereafter the nuclei were resuspended in 200 mL antibody buffer containing 1:50 anti-Flag Ab (Sigma-Aldrich, F1804-200UG) for the BG4 samples or 2 ug anti-Rabbit Ab (G4 CUT&Tag: Antibodies Online, ABIN101961; HA CUT&Tag: EpiCypher, 13-0047) and incubated for 1 hr at room temperature with end-over-end rotation. For the BG4 samples an additional antibody incubation followed three washes by resuspending the nuclei in 200 mL antibody buffer with 1:50 anti-mouse Ab (Sigma-Aldrich, M7023) and incubating for 1 hr at room temperature. The nuclei were then washed three times and resuspended in 200 mL dig-300 wash buffer (20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 0.01% digitonin, 0.01% NP40 and Roche cOmplete Protease Inhibitor EDTA-free) with 2.5 μL pA-Tn5 (EpiCypher, 15-1017) and incubated at room. After three washes with dig-300 wash buffer with centrifugation at 300 x g for 6 min, the nuclei were resuspended in 200 mL tagmentation buffer (dig-300 wash buffer containing 10 mM MgCl2) and incubated at 37°C for 1 hr. The reaction was then stopped by adding 6.7 mL of 0.5 M EDTA, 2 mL of 10% SDS and 5 mL of 20 mg/mL proteinase K and incubating at 63°C for 1 hr. Genomic DNA was purified using a DNA Clean & Concentrator-5 kit (Zymo Research, D4014), eluted in 20 mL, and RNA was digested by adding 1 mL RNase cocktail (Fisher Scientific, AM2288) with incubation for 20 min at 37°C. For the library prep, 2 uL of 10 uM uniquely barcoded universal i5 and i7 primers as well as 25 uL NEBNext Ultra II Q5 2x PCR Master mix (NEB, M0544L) were added to the 21 uL DNA and a short PCR was performed (72°C for 5 min, 98°C for 30 sec, 13 x (98°C for 10 sec, 63°C for 30 sec), 72°C for 1 min). The library PCR products were cleaned up with Agencourt AMPure XP beads (Beckman Coulter, A63881)