GSM6217079: HEK293T, H4S47A, NS_rep1; Homo sapiens; OTHER
HEK293T cells were collected and lysed with DNAzol (Invitrogen, 10503027) at room temperature for 5 min. Genomic DNA with 1% spike-in mouse DNA (R1 cells) was precipitated with 100% ethanol, denatured by boiling for 10 min at 100 °C and then loaded onto 40 mL neutral 5-30% sucrose gradients prepared in TEN500 (10 mM Tris pH 7.9, 2 mM EDTA, 500 mM NaCl) in ultra-clear tubes. Gradients was centrifuged in SW28 rotor at 25,000 rpm for 20 h. A 1 mL-fraction was withdrawn from the top of the gradients. DNA fragments ranging from 0.5-2.5 kb were collected and purified with ethanol. Half of the total purified DNA was treated with RNase A. Subsequently, all DNA was treated with lambda exonuclease (NEB, MO262L) three times and purified with Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare, 28903470). Using Klenow Fragment (NEB, M0212L) and random primers (Invitrogen, 48190011), ssDNA was converted to dsDNA and then subjected to DNA library preparation using Next Ultra DNA Library Prep Kit (NEB, E7645) according to the manufactory's instructions. DNA library preparation using Next Ultra DNA Library Prep Kit (NEB, E7645) according to the manufactory's instructions.