GSM5808526: G2-M_HEK293T-LiveG4ID_LowDox_LiveG4ID-seq_rep3; Homo sapiens; OTHER
Purified genomic DNA from LiveG4ID-seq were mixed with the universal i5 primer and barcoded i7 primer, and amplified with 5X Ampli PCR Mix (Novoprotein, Shanghai). The library PCR products were cleaned up with DNA Clean Beads (Vazyme, Nanjing). The first antibody for the identification of chromatin G-quadruplexes was expressed as LiveG4ID in living cells. Then LiveG4ID-seq experiment was performed according to a protocol similar to CUT&Tag. Briefly, 20 K cells were harvested, washed with wash buffer (20mM HEPES pH 7.5, 150mM NaCl, 0.5mM spermidine), and immobilized to concanavalin A–coated beads (Novoprotein, Shanghai) with incubation at room temperature for 10 min. The beads-bound cells were incubated in 50 μl of buffer (wash buffer with 2mM EDTA, 0.1% BSA and 0.05% digitonin) with 1:100 Rabbit anti-GFP antibody (Yeasen, Shanghai) dilution at RT by rotating for 2 h. Then the Rabbit anti-GFP antibody buffer was removed. The incubated cells were then resuspended with 100 ul of dig-wash buffer with 1:200 dilution of Goat anti-Rabbit IgG antibody (Abcam, ab6702) and incubated at room temperature for 1 hour by rotating. After wash with 500 uL dig-wash buffer twice, cells were resuspended in 60 ul of dig-300 buffer (20mM HEPES pH 7.5, 300mM NaCl and 0.5mM spermidine, 0.01% digitonin) with 1:100 dilution of pA/G-Tn5 transposome and incubated at room temperature for 1 hour by rotating. pA/G-Tn5-bound cells were washed with 500 ul of dig-300 buffer twice, followed by tagmentation in 50ul of tagmentation buffer (dig-300 buffer with 10mM MgCl2) at 37℃ for 1 hour. After tagmentation, 1 uL of 10% SDS was added and further incubated at 55℃ for 10 min to stop tagmentation and detach Tn5 from DNA. The DNA fragments were extracted by 3:1 v/v Tagment DNA Extract Beads (Novoprotein, Shanghai).