title: GSM5397864: SGBS_day4_batch2_rep4; Homo sapiens; ATAC-seq
library_construction_protocol: All extraction steps were performed at 4C. For SGBS, nuclei were extracted by pelleting 50,000 cells at 500 RCF, resuspended and incubated on ice 3 minutes in 50 ul buffer containing 10 mM Tris-HCL, 10 mM NaCL, 3 mM MgCl2, 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin. 1 mL buffer containing 10 mM Tris-HCL, 10 mM NaCL, 3 mM MgCl2, and 0.1% Tween-20 was added. Nuclei were pelleted at 500 RCF for 10 minutes. For adipose tissue, 50 mg of frozen tissue was crushed and then homogenized in a dounce with 1 mL buffer containing 20mM Tris-HCl, 50mM EDTA, 5mM Spermidine, 0.15mM Spermine, 0.1% mercaptoethanol, and 40% Glycerol, pH 7.5. The homogenized sample was added to 9 mL of the same buffer and incubated on ice for 5 minutes and filtered through a Miracloth before pelleting at 1100xg for 10 minutes. The pellet was resuspended in 200 uL of the same buffer with an addition of 0.5% Triton X-100 and centrifuged at 500xg for 5 minutes. The pellet was resuspended in 250 uL of a wash buffer containing 10mM Tris-HCl, 10mM NaCl, 3mM MgCl2, pH 7.4. For both SGBS and adipose tissue, nuclei were suspended in 25 μl transposase buffer from the Nextera kit, 5 μl TDE1 Transposase enzyme, and 20 μl water for 30 minutes at 37C. Transposition reaction was cleaned with a Zymo DNA Clean and Concentrator kit. Libraries were amplified with 10 μl transposed DNA, 10 μl nuclease-free H2O, 2.5 μl 25 μM PCR Primer 1, 2.5 μl 25 μM Barcoded PCR Primer 2, and 25 μl NEBNext High-Fidelity 2X PCR Master Mix. Final PCR product was cleaned again with Zymo DNA Clean and Concentrator kit.