GSM8129284: Primed-H9_ATACseq_rep3; Homo sapiens; ATAC-seq
the sample used to perform RNA-seq, ATAC-seq, CUT&Tag-seq are prepared based on the commonly used method. AS for ATAC-seq, Cells were harvested and counted. Number of 500~1000 cells were used for ATAC-seq. The number of cells at this step is crucial, as the transposase-to-cell ratio determines the distribution of DNA fragments generated. Sample with 500~ 1000 cells was centrifuged for 5 min at 500 × g at 4 °C. Supernatant was removed and discarded. Cells were washed once with 50 μl of cold PBS buffer, centrifuged for 5 min at 500 × at 4 °C, and supernatant removed and discarded. The cell pellet in 50 μl of cold lysis buffer was resuspended by gently pipetting up and down and lysed for 10 min on ice, followed by centrifugation immediately for 10 min at 500 × g at 4 °C. The supernatant was discarded, and immediately continued to transposition reaction. Lysis buffer used was, 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40.For transposition reaction and purification, TruePrep® DNA Libraryaf Prep Kit V2 for Illumina (Vazyme, TD502, 5ng) for tagmentation was used. After tagmentation reaction, AMP XP beads for purifying was used. In preparation, AMPure XP beads were equilibrated at room temperature for 30 minutes and mixed well. 25 μl (0.6x) beads were pipetted into the 50 μl PCR product, gently pipetted and mixed, and incubated at room temperature for 5 min (to remove long fragments). The reaction tube was centrifuged briefly and placed on a magnetic stand. After clarification, the supernatant (75 μl) was transferred to a new tube (be careful to mark the tube cap and tube body), and the magnetic beads discarded. 60 μl (1.2x) beads were pipetted into the supernatant, mixed by pipetting, and incubated at room temperature for 5 min (magnetic beads can be added to the new EP tube during the first incubation). The reaction tube was centrifuged briefly and placed on a magnetic stand. After clarification, the supernatant was removed, the tube was kept on the magnetic stand.  200 μl of freshly prepared 80% ethanol was added to rinse the magnetic beads, incubated at room temperature for 30 s, and the supernatant carefully removed. The rinse was repeated three times. The tube was kept on the magnetic stand, the lid left open and dry (not too dry, no alcohol residue at the bottom of the tube). The tube was taken out of the magnetic stand, 25 μl of sterile ultrapure water added, the magnetic beads smashed from the wall, mixed well, and incubated at room temperature for 5 min. The reaction tube was dentrifuged briefly and placed on a magnetic stand. After clarification, 20 μl of supernatant was carefully transferred to a new tube and stored at −20°C.Library was constructed as follow. TruePrep TM Index V4 for Illumina (Vazyme TD204) was used to enrich DNA and PCR amplification. The PCR products were purified with VAHTS DNA Clean Beads (Vazyme N411). At last, DNA fragments of preferentially about 300 bp in length were selected, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA)