title: GSM6696928: POLE3-/- H3.3 D0 ESC CUT&Tag repeat 2; Mus musculus; OTHER
library_name: GSM6696928
library_construction_protocol: 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 μL) (Beckman Counter), and libraries were incubated with beads for 10 min at RT, washed twice gently in 80% ethanol, and eluted in 20 μL EB buffer. The libraries were used for paired-end (2×150 bp) sequencing on an Illumina Novaseq or HiSeq Xten platform by Annoroad Gene Technology Co. Ltd. RNA-seq library preparation and deep sequencing were performed by Annoroad Gene Technology Co. Ltd.. Cell suspensions were barcoded through the 10x Chromium Single Cell platform using Chromium Single Cell 3′ Library, Gel Bead and Multiplex Kit (10x Genomics).