GSM8746488: ATAC-seq, mESC-Pou5F1-lin-del, Clone1, rep1; Mus musculus; ATAC-seq
50,000 of freshly harvested cells were resuspended in ice-cold RSB buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2). The suspension was centrifuged at 500xg for 3 min using a refrigerated centrifuge. The cell pellet was washed once with 50 μl ice-cold PBS and centrifuged at 500xg for 3 min at 4°C. The cell pellet was resuspended in ice-cold Lysis Buffer (48.5 μl RSB buffer supplemented with 0.5 μl of 10% NP-40, 0.5 μl of 10% Tween-20, 0.5 μl of 1% Digitonin) and kept on ice for 3 min. Then, 1 ml of wash buffer (RSB buffer supplemented with 0.1% Tween-20) was added on the lysed cells. Nuclei were pelleted at 500xg for 10 min at 4°C and the supernatant (cytoplasm fraction) was carefully discarded. The nuclei were resuspended in the transposition mix (25 μl of 2X Tagment DNA Buffer (Illumina, #20034197), 16.5 μl of 1X PBS, 0.5 μl 10% of Tween-20, 0.5 μl of 1% Digitonin, 5 μl nuclease-free H2O). Next, 2.5 μl of Tn5 Transposase (Illumina, #20034197) was added to the reaction mix by pipetting up and down 6 times. The reaction was then incubated at 37℃ for 30 minutes on a thermomixer at 1,000 rpm. Finally, the tagmented DNA was isolated using Qiagen MinElute Reaction Cleanup Kit (Qiagen, # 28204) following the manufacturer's instructions. ATAC-seq libraries were generated by PCR amplification using custom-made barcodes. Libraries were amplified with 7-10 cycles using NEBNext High-Fidelity 2X PCR Master Mix (NEB, #M0541S). To remove primer dimers and DNA fragments shorter than 150bp, libraries were purified with SpeedBeads beads. 1.8X volume of SpeedBeads beads (81 μl beads for 45 μl PCR reaction) was used to clean PCR amplified DNA library. The quality of the DNA libraries was assessed using Agilent HS DNA Screen Tape (Agilent. Santa Clara, CA). The concentration of the DNA libraries was measured by Qubit Fluorometric Quantitation. Equimolar DNA libraries were pooled to a final concentration of 2 nM. The ATAC-seq libraries were sequenced by Novagene (Novogene, UK) on Illumina HiSeq 6000 using pair-ends PE150 in biological duplicates (Illumina, San Diego, CA).