GSM5501207: K562_50k_bg4_cellNO_rep1_tec2; Homo sapiens; OTHER
Bulk G4 CUT&Tag: Harvested cells were fixed in 0.1% formaldehyde in PBS for 2min at room temperature and quenched with glycine at a final concentration of 0.075M. Fixed cells were then resuspended in wash buffer (20mM HEPES pH7.5, 150mM KCl, 0.5mM spermidine in nuclease-free water with a Roche Complete Protease Inhibitor EDTA-free tablet) and cell concentration was measured again for cellNO samples at this stage. 100µL of the cell suspension was mixed with 10µL pre-washed concanavalin A beads (in 20mM HEPES pH7.5, 10mM KCl, 1mM CaCl2, 1mM MnCl2 in nuclease-free water) at 25°C for 10min at 600rpm. Following wash steps were performed using magnetic rack. Bead-bound fixed cells were washed twice with wash buffer and then incubated in 50µL antibody buffer (2mM EDTA, 0.1% BSA, 0.05% digitonin in wash buffer) with 4µL of 5.4µM BG4 scFv (no primary antibody was added for IgG control samples) at 25°C for 2h at 600rpm. Next, cells were washed twice with 100µL dig-wash buffer (0.05% digitonin in wash buffer) and then incubated in 50µL dig-wash buffer with 2µL of anti-flag antibody (Cell Signaling Technology Cat# 2368, RRID:AB_2217020) at 25°C for 1h at 600rpm. After that, cells were washed three times with 500µL dig-wash buffer and then incubated in 50µL dig-wash buffer with 0.5µL of anti-rabbit antibody (Antibodies-Online Cat# ABIN101961, RRID:AB_10775589) at 25°C for 1h at 600rpm. Cells were washed three times with 500µL dig-wash buffer and then incubated in 1:250 dilution of pA-Tn5 in 50µL Dig-300 buffer (20mM HEPES pH7.5, 300mM KCl, 0.5mM spermidine, 0.01% digitonin in nuclease-free water with a Roche Complete Protease Inhibitor EDTA-free tablet) at 25°C for 1h at 600rpm. To activate tagmentation, cells were washed three times with 500µL Dig-300 buffer and then incubated in 300µL tagmentation buffer (10mM MgCl2 in Dig-300 buffer) at 37°C for 1h at 600rpm. After tagmentation, the cells were washed twice with 500µL TAPS wash buffer (10mM TAPS, 0.2mM EDTA in nuclease-free water). 100µL of extraction buffer (0.5mg/mL proteinase K, 0.5% SDS in 10mM Tris-HCl pH8.0) was added to each sample, mixed by vortexing, and incubated at 55°C for 1h at 800rpm. Tagmented DNA was purified through phenol/chloroform extraction and ethanol precipitation. The pellet was dissolved in 25µL elution buffer 1 (10mM Tris-HCl pH8, 1mM EDTA, 1/400 RNAseA in nuclease-free water) by vortexing and incubated at 37°C for 10min at 800rpm. In each 0.2mL PCR tube, 25µL NEBNext HiFi 2x PCR master mix, 2µL of 10µM Ad1, 2µL of 10µM Ad2 [Buenrostro, J.D. et al. Nature (2015)] and 21µL tagmented DNA were added. The PCR profile was as follow: 72°C for 5 min, 98°C for 30s, followed by 10 cycles of 98°C for 10s and 63°C for 10s, and one cycle of 72°C for 1 minute. Libraries were purified using 1.3X Ampure XP beads and eluted 25µL 10mM Tris-HCl for 5min at room temperature. Pooled libraries were size selected and concentrated using Ampure XP beads and eluted in 10mM Tris-HCl. The libraries were sequenced on a Illumina NextSeq 500 sequencer with a paired-end format of 36 bp x 2 using the High Output kit.