title: GSM8170807: ATAC-seq on  GFP-dTAG-Ctcf ESC, dTAG13 100nM treatment for 3hrs, replicate2; Mus musculus; ATAC-seq
library_construction_protocol: Cells were washed once in pre-warmed PBS, disaggregated using TrypLE (Thermo Fisher Scientific), and trypsinization was stopped by adding cold media. Two hundred thousand cells were transferred to LoBind microfuge tube (Eppendorf) and centrifuged at 500g for 5min at 4ºC. After centrifugation, the cells were suspended in buffer A (10mM HEPES pH 7.9, 10mM KCl, 1.5mM MgCl2, 0.34M Sucrose, 10% Glycerol, 10% Triton-X), mixed by tapping and incubated on ice for 7min. After centrifugation, buffer A was removed, and nuclei were suspended in lysis buffer (10mM Tris-HCl pH7.4, 10mM NaCl, 3mM MgCl2, 0.3% Igepal). A quarter of nuclei was transferred to a new Lobind tube, briefly vortexed, incubated on ice for 15min, vortexed briefly again, and pelleted down at 600g for 10min at 4ºC. After removing lysis buffer, nuclei were recovered with 2.5 μL 2X TD buffer and 2.5 μL transposase (TDE1) mixture (Illumina). After pipetting several times, the nuclei were incubated for 30min at 37 ºC on a rocking mixer. The transposed DNA was purified using the MinElute PCR Purification kit (Qiagen) following the manufacture's protocol. The transposed genomic region was PCR-amplified using Nextera Primers ( https://www.encodeproject.org/documents/860c95a8-fdc4-46bd-b9b5- d44242ff20ef/@@download/attachment/ATAC_garberlab_protocol.pdf ) and NEBNext High-Fidelity 2x PCR Master Mix (NEB). PCR product was purified using Agencourt® AMPure® XP beads (Beckman Coulter) and sequenced on a Next-Seq 2000 Sequencer (Illumina).