title: GSM7778542: HEK293T, EMX1, 200928, Rep1; Homo sapiens; ATAC-seq
library_construction_protocol: The Dynabeads™ M-280 sheep anti-Rabbit IgG (Invitrogen, 11201D) was diluted at 1:10 in 1× PBS (Invitrogen, AM9624) to prepare 50 µL solution at room temperature. The mixture was placed on DynaMag™-96 Side Skirted (Invitrogen, 12027) for 3 minutes, and the supernatant was carefully pipetted off. 1 µL of Nesprin1 antibody (abcam, ab192234) and 1× PBS was added to a volume of 50 µL, and it was rotated at room temperature for 30 minutes. The tube was then transferred onto the magnetic strand for 3 minutes, the supernatant was pipetted off, and the beads were resuspended in 5µL of 1× PBS. This washing step with 1× PBS was repeated. The beads were subsequently resuspended with 250 µL (10µL per sample) of cell lysis buffer, which containing 10 mM HEPES (Gibco, 15630106), 2.5 mM MgCl2 (Invitrogen, AM9530G), 10 mM KCl (Sigma, P9333), 1mM DTT (ThermoFisher, 707265ML), 0.5% Triton™ X-100 (Sigma, 93443), 2 U Recombinant RNase Inhibitor (Takara, 2313A).500 GFP-positive cells were sorted into each well of a 96-well plate containing 10 µL lysis buffer by flow cytometer (BD Influx, 14005297) . The plate was centrifuged at 700 g for 1 minute, and then incubated at  4°C and 300 rpm for 30 minutes. After placing the plate on DynaMag™-96 Side Skirted for 3 minutes, the supernatant was carefully transferred to another plate for RNA-seq. The tagmentation mix containing TTE MIX (Vazyme, TD501), 5× TTBL (Vazyme, TD501), 0.3× PBS, 0.1%  Tween-20 (Sigma, P9416), 0.1% IGEPAL-630 (sigma, 13021), 10% DMF(Sigma, D4551) and 0.01% Digitonin (Promega, G9441), was prepared before this transfer. 5 µL tagmentation mix was used to resuspend the nuclei by vortexing for 1 s. Then, the nuclei were incubated at a prewarmed 37°C for 30 minutes. 5 µL of stop mix (1% SDS (Invitrogen, AM9822), 100 mM EDTA (Invitrogen, AM9260G) 0.4 µg/µl Proteinase K (NEB, P8107S, 0.2 µg/µl Carrier RNA (Applied Biosystems™, 4382878)) was then added and incubated at 55°C for 30 minutes. After that, 22 µL of VAHTS DNA Clean Beads (Vazyme, N411-02) was added, and the mixture was left at room temperature for 10 minutes. It was subsequently washed with 80% Ethanol (Sigma, E7023) twice. The DNA fragment was eluted in 10 µL UltraPure DNase/RNase-Free Watre（Invitrogen, 10977015）, and 12.5 µL 2× KAPA HIFI ready mix (Roche, KK2602) and 1.25 µL 25 µM barcode primer were added for PCR amplification. The PCR program was set as follow: 72°C for 5 minutes, 98°C for 3 minutes, followed by 15 cycles of 98°C for 20 s, 63°C for 30 s, and 72°C  for 3 minutes, and then hold at 4°C until further use.RNA was purified by 2.2× VAHTS RNA Clean Beads (Vazyme, N412-03) from the supernatant and reverse transcribed using the SMART-seq2 protocol (Picelli S, 2014). The RNA-seq library construction was done using TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD501), following the manufacturer's manual.