GSM9051174: IgG_WT_B1; Homo sapiens; OTHER
Sequencing libraries were generated by mixing 25 uL NEBNext High Fidelity 2X PCR Master Mix (M0541, New England Biolabs), 2 uL of P5 indexing primer (10 mM), 2 uL of P7 indexing primer (10 mM) with 21 uL of purified DNA in a PCR reaction under the following conditions: 72 ºC for 5 min, 98 ºC for 30 sec followed by n cycles of 98 ºC 10 sec, 63 ºC for 10 sec, and a final extension at 72 ºC for 1 min. For BG4 and SG4 libraries n=8 cycles were used, for all other CUT&Tag libraries n=10 was used. Libraries were purified using Ampure XP beads (A63882, Beckman Coulter Life Sciences) by incubation with 0.4X ratio of beads for 15 min, followed by supernatant incubation with 1.4X ratio of beads for 15 min. Beads were washed twice with 800 mL 80% ethanol and eluted in 25 uL of 10mM Tris-HCl pH 8.0. Library size distribution was analysed on a Tapestation 2200 (Agilent Technologies) and library concentrations were quantified by NEBNext Library Quant Kit for Illumina (E7630, New England BioLabs). Individual libraries were pooled for optimal read balancing and sequenced paired end with NextSeq 2000 P2 or P3 Reagents 100 Cycles v3 (20046811, 20040559, Illumina, Inc.) on the NextSeq 2000 platform (Illumina, Inc.). Dynabeads MyOne Streptavidin T1 beads (65601, Thermo Fisher Scientific) were incubated with Concanavalin A (C2272, biotin conjugate, Sigma-Aldrich) in ratio of 10 mg/mL beads to 1.15 mg/mL Concanavalin A at 22 ºC for 30 min at 400 rpm on a thermoshaker (Eppendorf). Volume sufficient for 10 µL beads per reaction was prepared. Beads were washed with 1 mL of binding buffer (20mM HEPES pH 7.5, 10mM KCl, 1mM CaCl2, 1mM MnCl2) and resuspended in volume of binding buffer sufficient for 10 uL beads per reaction. Cells were fixed in 0.1% formaldehyde (28906, Thermo Fisher Scientific) in 1X PBS for 2 min at room temperature and quenched with 0.075M glycine. Cells were centrifuged at 1300xg 4 ºC for 4 min and resuspended in wash buffer (20mM HEPES pH 7.5, 150mM KCl, 0.5mM spermidine (S0266, Sigma-Aldrich), one tablet protease inhibitor (11873580001, Roche cOmplete, EDTA-free)) at 500 cells/mL. 10 uL beads were incubated with 100 uL of cell suspension at 25 ºC for 10 min at 600 rpm, washed twice with 100 uL wash buffer and resuspended in 50 uL of antibody buffer (2mM EDTA, 0.1% BSA (A8577, Sigma-Aldrich), 0.05% digitonin (300410, Merck Millipore) in wash buffer). Primary antibody was added and incubated at 4 ºC at 600rpm overnight. Primary antibodies used include; BG4 scFv (made in house) at 0.5 mM per reaction, SG4 (made in house) at 1 mM per reaction, anti-H3K4me1 antibody (710795, Thermo Fisher Scientific), anti-H3K27ac antibody (ab4729, Abcam), anti-Ser5 phosphorylated Rpb1 antibody (#13523, Cell Signaling Technology), anti-GATA1 antibody (ab181544, Abcam), anti-BRG1 antibody (ab110641, Abcam), anti-P300 antibody (sc-48343, Santa Cruz Biotechnology), Normal Rabbit IgG (2729, Cell Signaling Technology)). Cells were washed twice with 100 uL dig wash buffer (0.05% digitonin in wash buffer) and resuspended in 50 uL of dig-wash buffer. Secondary antibody was then added and incubated at 25 ºC at 600 rpm for 1 hr. For BG4 and SG4 samples, 2 uL of rabbit anti-FLAG antibody (#2368, Cell Signaling Technology) was added, for all other rabbit primary antibodies 0.5 uL of Guinea Pig anti-rabbit IgG antibody (ABIN101961, antibodies-online.com) was added, and for mouse primary antibodies 0.5 uL of Rabbit Anti-mouse IgG H&L (ab46540, Abcam) was added per reaction. Cells were then washed three times with 500 uL dig-wash buffer and resuspended in 50 uL dig-300 buffer (20mM 20mM HEPES pH 7.5, 300mM KCl, 0.5mM spermidine, 0.01% digitonin, one tablet protease inhibitor). pA-Tn5 (2 mM, made in house) was added in 1:250 dilution to each sample and incubated at 25 ºC at 600 rpm for 1 hr. Cells were washed three times with 500 uL dig-300 buffer and resuspended in 300 uL tagmentation buffer (10mM MgCl2 in dig-300 buffer) and incubated at 37 ºC at 600 rpm for 1 hr. Cells were then washed twice with 500 uL TAPS wash buffer (10 mM TAPS (J63268.AE, Thermo Fisher Scientific) and resuspended in 100 uL of proteinase K solution (0.5 mg/mL Proteinase K (EO0491, Thermo Fisher Scientific), 0.5% SDS in 10mM Tris-HCl pH 8.0). Samples were vortexed on full and incubated at 55 ºC for 1 hr at 800 rpm. DNA was extracted using DNA Clean & Concentrator 5 (D4013, Zymo Research) following the manufacturer's instructions, DNA was eluted in 25 uL DNA elution buffer.