title: GSM9444769: ANSC_H9_rep1; Homo sapiens; ATAC-seq
library_construction_protocol: Utilizing the Hyperactive ATAC-Seq Library Prep Kit for Illumina (Vazyme TD711) for cell lysis and DNA extraction, cells were first digested with 0.25% trypsin at room temperature, then collected and counted. A total of 100,000 cells were resuspended in 50 μl of pre-chilled TW Buffer and centrifuged at 4°C, 2,300 rpm for 5 minutes, after which the supernatant was discarded. This washing process was repeated twice. The cells were then resuspended in 50 μl of pre-chilled Lysis Buffer, placed on ice for 5 minutes, and centrifuged again at 4°C, 2,300 rpm for 10 minutes, with the supernatant discarded. To each sample, 50 μl of pre-chilled Fragmentation Mix was added, and the mixture was gently pipetted 10 times to ensure homogeneity. The samples were then incubated at 37°C for 30 minutes. Post-reaction, the EP tubes were briefly centrifuged, and the contents were gently mixed by pipetting and transferred to a 200 μl PCR tube. Immediately after transfer, 5 μl of Stop Buffer was added to each sample, mixed gently by pipetting 10 times, and allowed to stand at room temperature for 5 minutes. The magnetic beads were equilibrated to room temperature, vortexed to mix the ATAC DNA Extract Beads, and 100 μl was added to 55 μl of the terminated sample, mixed thoroughly by pipetting 10 times, and incubated at room temperature for 5 minutes. The reaction tubes were briefly centrifuged and placed on a magnetic stand to separate the magnetic beads from the liquid, and the supernatant was carefully removed after approximately 5 minutes of clarification. The magnetic beads were washed twice with 200 μl of freshly prepared 80% ethanol, incubated at room temperature for 30 seconds, and the supernatant was carefully removed. The reaction tubes were kept on the magnetic stand and air-dried with the lid open for approximately 5 minutes. The tubes were then removed from the magnetic stand, and 26 μl of Nuclease-free ddH2O was added to elute the DNA, mixed thoroughly by pipetting 10 times, and incubated at room temperature for 5 minutes. Finally, the tubes were briefly centrifuged and placed on the magnetic stand to separate the magnetic beads from the liquid, and after approximately 5 minutes of clarification, 24 μl of the supernatant was carefully transferred to a new sterile PCR tube for direct proceeding to the next PCR amplification step. The ATAC DNA Clean Beads were vortexed thoroughly, and 33 µl (0.55×) was transferred to 60 µl of PCR product. The mixture was homogenized by vortexing or pipetting 10 times and incubated at room temperature for 5 min. The reaction tube was briefly centrifuged and placed on a magnetic stand to separate the beads from the solution. After the supernatant cleared (~5 min), 88 µl of the supernatant (supernatant volume = total volume 93 µl – 5 µl) was carefully transferred to a new sterile PCR tube, and the beads were discarded. The ATAC DNA Clean Beads were vortexed again, and 72 µl (1.2×) was added to the supernatant, followed by thorough mixing via vortexing or pipetting 10 times and incubation at room temperature for 5 min. The tube was briefly centrifuged and placed on the magnetic stand until the solution cleared (~5 min), after which the supernatant was removed. While keeping the tube on the magnetic stand, the beads were washed twice with 200 µl of freshly prepared 80% ethanol, incubating for 30 sec each time before carefully removing the supernatant. The tube remained on the magnetic stand with the lid open for ~5 min to air-dry the beads. The tube was then removed from the stand, and 22 µl of nuclease-free ddH₂O was added for elution. The mixture was vortexed or pipetted 10 times to ensure complete resuspension and incubated at room temperature for 5 min. After brief centrifugation and placement on the magnetic stand, the cleared supernatant (~5 min) was collected, and 20 µl of the eluate was transferred to a new sterile PCR tube for storage at –30 to –15°C.