title: GSM8059392: HEK293T_BG4_CUT&Tag_hg19_rep1; Homo sapiens; OTHER
library_construction_protocol: 2 × 105 cells were treated with NE1 buffer (20 mM HEPES-KOH pH7.9, 10 mM KCl, 0.5 mM Spermidine, 0.1% (v/v) Triton X-100, 20% (v/v) Glycerol ) on ice for 10 minutes, then fixed with 0.1% formaldehyde in PBS for 2 min and harvested. The cells were washed with PBS, and immobilized to Concanavalin A Magnetic Beads (Beyotime, P2156) with incubation at room temperature for 10 min. The bead-bound cells were incubated in 200 μL of primary antibody buffer (20 mM HEPES-KOH pH7.5, 150 mM NaCl, 0.5 mM Spermidine, 1% (v/v) BSA, 2 mM EDTA) with 40 μl 0.05 mg/mL FLAG-tagged BG4 antibody or 2 μL 1 mg/mL Mouse IgG Isotype Control (Share-bio, SB-A00001) antibody at 4 ℃ by rotating overnight. The next day, Flag-Tag Mouse Monoclonal Antibody (Share-bio, SB-AB0008), Rabbit Anti-Mouse IgG H&L antibody (Bioss, bs-0296R), pA-Tn5 adapter complex were incubated at 37 ℃ for 1 hr. All three antibodies are diluted 1:200. Flag-Tag Mouse Monoclonal Antibody and Rabbit Anti-Mouse IgG H&L antibody were diluted with BSA-wash buffer (20 mM HEPES-KOH pH7.5, 150 mM NaCl, 0.5 mM Spermidine, 1% (v/v) BSA), and pA-Tn5 adapter complex was diluted with BSA-300 buffer (20 mM HEPES-KOH pH7.5, 300 mM NaCl, 0.5 mM Spermidine, 1% (v/v) BSA). After each incubation of antibodies, wash 3 times with the corresponding antibody incubation solution for 5 min. Tagmentation was performed with 50 μL Tagmentation buffer in 37℃ for 1 hr (20 mM HEPES-KOH pH7.5, 300 mM NaCl, 0.5 mM Spermidine, 1% (v/v) BSA, 10 mM MgCl2). After tagmentation, 1 μL 500 mM EDTA, 1 μL 20 mg/mL proteinase K and 1 μL 10% (w/v) SDS were added and further incubated in 55 ℃ for 1 hr and 70℃ for 20 minutes to stop tagmentation and digest protein. 122 μL DNA Selection Beads (Yeason, 12601ES) were added to the supernatant of the aforementioned process. Wash twice in 80% ethanol and elute the DNA using 35 μL TE buffer. To generate G4 libraries, purified genomic DNA was amplified with the universal i5 primer and barcoded i7 primer using NEBNext Ultra II Q5 Master Mix (NEB, M0544). The library PCR products were electrophoresed using 2% (w/v) agarose gel and 200-500 bp size fragments were recovered with Gel Extraction Kit (Sangon Biotech, B511139). Sequencing were conducted on an Illumina Novaseq-PE150 instrument. Cleavage under targets and tagmentation (CUT&Tag)