GSM5501223: MCF7_10k_bg4_10X_5th_ref_rep1_tec1; Homo sapiens; OTHER
Bulk G4 CUT&Tag (single nuclei reference): 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. Nuclei were extracted by incubating the fixed cells in 1mL NE1 buffer (20mM HEPES pH7.5, 10mM KCl, 0.5mM spermidine, 0.1% Triton X-100 and 20% glycerol in nuclease-free water with a Roche Complete Protease Inhibitor EDTA-Free tablet) for 10min on ice, washed once with 1mL PBS and resuspended in 500µL 1% BSA antibody buffer (2mM EDTA, 1% BSA, 0.05% digitonin, 20mM HEPES pH7.5, 150mM KCl, 0.5mM spermidine in nuclease-free water with a Roche Complete Protease Inhibitor EDTA-free tablet). Following wash steps were performed through centrifugations at 4°C for 3min at 600×g in a swinging bucket rotor for the initial steps, and then at 4°C for 3min at 300×g after third antibody incubation step when the nuclei were in Dig-300 buffer. 500,000 fixed nuclei were transferred to 0.2mL Lo-retention tubes and washed twice with 200µL 1% BSA antibody buffer, resuspended in 50µL 1% BSA antibody buffer and incubated at 25°C for 1h at 600rpm. 2µL of 5.4µM BG4 scFv was added to each sample and incubated in cold room overnight at 600rpm. After that, cells were washed twice with 100µL dig-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. Next, cells were washed twice with 100µL 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 100µL 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) and then incubated in 1:250 dilution of pA-Tn5 in 50µL Dig-300 buffer at 25°C for 1h at 600rpm. To activate tagmentation, cells were washed three times with 100µL Dig-300 buffer and incubated in 100µL tagmentation buffer (10mM MgCl2 in Dig-300 buffer) at 37°C for 1h in thermocycler and mixed every 10min. After tagmentation, BSA was added to the samples to a final concentration of 1% and centrifuged at 4°C for 3min at 600×g. The nuclei were washed twice with 1X diluted nuclei buffer with 1% BSA by centrifugation at 4°C for 3min at 600×g and resuspended in 25µL 1X diluted nuclei buffer on ice. Then, the nuclei were checked and counted by staining with trypan blue and observed under microscope using hemocytometer. 100µL of extraction buffer (0.5mg/mL proteinase K, 0.5% SDS in 10mM Tris-HCl pH8.0) was added to 10,000 tagmented nuclei, mixed by vortexing, and incubated at 55°C for 1h at 800rpm. Tagmented DNA was purified using 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 (NEB, M0541), 2µL of 10µM Ad1, 2µL of 10µM Ad2 [Buenrostro, J.D. et al. Nature (2015)] and 21µL 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.