GSM5609902: MCM2-2A H3K27me3 eSPAN 24h; Mus musculus; OTHER
GSM5609902
CUT&Tag was performed as previously reported with modifications4,5. Cells (1-2×105) were washed twice in 1 mL Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1×Protease inhibitor cocktail) at room temperature. Concanavalin-coated magnetic beads (Bangs Laboratories) were washed twice in 1 mL Binding Buffer (20 mM HEPES pH 7.9, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2). Cells were resuspended in 1 mL Wash Buffer and incubated with 10 μL pre-washed Concanavalin-coated magnetic beads at room temperature for 15 min. Beads (bound to cells) were incubated in Antibody Buffer (Wash Buffer, 0.08% Digitonin for ESCs/0.05% Digitonin for other cells, 2 mM EDTA, 0.1% BSA, 1:100 dilution of the appropriate primary antibody) on a rotating platform overnight at 4°C. Prepare mixture of 1 μg appropriate secondary antibody (Table S1) and pAG-Tn5 adapter complex (Novoprotein) at 1:2 molar ratio at room temperature for 1 hour. Beads were washed once with 1 mL Dig-wash Buffer (Wash Buffer, 0.08% Digitonin for ESCs/0.05% Digitonin for other cells) and nutated in 100 μL Dig-300 buffer (20mM HEPES pH 7.5, 300mM NaCl, 0.5mM Spermidine, 1×Protease inhibitor cocktail, 0.01% Digitonin) added the secondary antibody bound pAG-Tn5 complex at room temperature for 1 hour. Beads were washed 3 times with 1 mL Dig-300 buffer and resuspended in Tagmentation Buffer (Dig-300 buffer, 10 mM MgCl2) at 37°C for 1 hour. Beads were washed once with 1 mL TAPS Wash Buffer (10mM TAPS, pH 7.5, 0.4 mM EDTA) and incubated in 250 µL Dig-300 buffer containing 15 mM EDTA, 0.1% SDS and 100 µg/mL Proteinase K at 37°C for 1 hour with shaking of 900rpm. Then CUT&Tag DNA was extracted by phenol-chloroform-isoamyl alcohol and purified again by DNA Clean & Concentrator-5 Kit (Zymo Research) to 20 µL EB buffer. CUT&Tag DNA was Incubated by RNase A (1/400) at 37°C for 10 min. To amplify libraries, 10 μL CUT&Tag DNA was mixed with 1 μL of a uniquely barcoded i5, 1 μL a uniquely barcoded i7 primer, using a different barcode for each sample, and 12 μL NEBNext 2× PCR mix, and run the following cycling conditions with lid heat on: 72 °C for 5 min (gap filling); 98 °C for 30 s; 14 cycles of 98 °C for 10 s and 63 °C for 10 s; final extension at 72 °C for 1 min and hold at 10 °C. Post-PCR clean-up was performed by adding 1× volume of Ampure XP beads (24 μ