GSM5553088: NCI-H660-ATAC; Homo sapiens; ATAC-seq
Cell pellet of 50,000 cells per sample was used for library preparation. Genomic DNA fragments for ATAC seq library was prepared as per protocol published in Buenrostro et al. 2013 and Chen et al. 2018 with some modifications. Briefly, cells were collected by incubating in trypsin for 5 minutes at room temperature and subsequent centrifugation at 2300 rpm for 5 minutes at 4°C. Cells were washed twice and re-suspended in PBS. 5 x 104 cell pellet was used for tagmentation by incubating in 50 µl of 1x THS-seq buffer (25µl 2x THS-buffer, 5µl 10x Digitonin, 2µl Illumina-TDE1 (Cat FC-121-1030) or ActiveMotif-Tn5 (Cat 53150)) for 20 minutes at 37°C with 800 rpm in ThermoMixer (Eppendorf). To stop the tagmentation reaction, equal volume of 2x Tagmentation Stop Buffer [10mM Tris-HCl (pH 8.0), 20mM EDTA (pH 8.0)] was added to reaction and incubated for 10 minutes on ice. For cell lysis, equal volume of 2x Lysis Buffer [100 mM Tris-HCl (pH8.0), 100 mM NaCl, 40 µg/ml Proteinase K, 0.4% SDS] was added to the tagmentation mix and incubated at 65°C for 15 minutes. The tagmented DNA library was purified in 20 µl EB using Qiaquick PCR purification kit (Qiagen). Library fragments were amplified using NEB Next High-Fidelity 2x PCR Master Mix (New England Biolab Cat #M0541) and custom Nextera PCR primers. Libraries were amplified for a total of 11 to 12 cycles and purified using Qiaquick PCR purification kit. Libraries were quantified by Qubit 1x dsDNA HS Assay kit (Cat Q33230), quality assessed by Agilent Bioanalyzer run using Agilent High Sensitivity Assay (kit no. 5067-4626) and sequenced using 75bp or 125bp Paired-end sequencing on a Illumina HiSeq 2500 following manufacturer's protocol.