title: GSM2065330: ATAC_77iSMA_F_rep3; Homo sapiens; OTHER
library_construction_protocol: The cell pellets were re-suspended in ice-cold cell lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630) supplemented with 1X protease inhibitor cocktail (Roche), incubated on ice for 5 min and centrifuged at 230 rcf for 5 min at 4°C. The supernatant was carefully removed and the nuclei were re-suspended in 25 µl of ice-cold 1X Tagment DNA Buffer (Illumina). 50,000 nuclei were used for the transposase reaction, which was carried out as described by Buenrostro et al. (Nat Methods. 2013 Dec;10(12):1213-8). The samples were purified with the DNA Clean & Concentrator-5 Kit (Zymo Research) and eluted with 20 µl of Elution Buffer (Qiagen). The samples were PCR-amplified using 25 µl of Nextera PCR Master Mix (Illumina), 5 µl of PCR Primer Cocktail (Illumina), 5 µl of Index primer 1 (i7, Illumina), and 5 µl of Index primer 2 (i5, Illumina). The following PCR reaction protocol was used: 3 min 72°C; 30 sec 98°C; 8 cycles (10 sec 98°C, 30 sec 63°C, 3 min 72°C). Samples were purified with the DNA Clean & Concentrator-5 Kit (Zymo Research), eluted with 20 µl of Elution Buffer (Qiagen), and loaded on 2% agarose gel (Invitrogen) for size-selection (175-250 bp). DNA was purified using the QIAquick Gel Extraction Kit (Qiagen) following the manufacturer's recommendation, and eluted with 20 µl of Elution Buffer (Qiagen). The DNA was further amplified with 1X NEBNext High-Fidelity PCR Master Mix (New England Biolabs), 200 nM of Primer 1 (5'-AATGATACGGCGACCACCGA-3'), and 200 nM of Primer 2 (5'-CAAGCAGAAGACGGCATACGA-3’). The following PCR reaction protocol was used: 30 sec 98°C; 4 cycles (10 sec 98°C, 30 sec 65°C, 30 sec 72°C); 5 min 72°C. The final libraries were purified using Agencourt AMPure XP beads (Beckman Coulter).
attributes.tag: GEO Accession
attributes.value: GSM2065330