GSM5242673: HEK293-PROSER1KO1D3-rep2-5hmC; Homo sapiens; OTHER
Genomic DNA of cells was extracted by using the QIAamp DSP DNA Mini Kit (Qiagen, 61304). 20 μg genomic DNA was sonicated to 200-600 bp by using a Bioruptor Plus sonication device (Diagenode, B01020001) in 300 μL 10 nM Tris-HCl pH 8.5 buffer. Set aside 100 ng fragmented DNA for use as input DNA. Set up a 100 μL reaction mixture containing 1 μg sonicated DNA, 1 μL provided Protease Inhibitor Cocktail, 10 μL provided Buffer C, and 4 μL provided 5-hydroxymethylcytidine pAb. Incubate overnight with end-to-end rotation at 4°C. Add 25 μL provided Protein G magnetic beads to each tube and incubate for 2 hours with end-to-end rotation at 4°C. Collect the beads by using the provide magnetic stand. Wash the beads three times with ice cold Buffer C and then two times with the ice cold provided Buffer D. Resuspend the washed beads with 50 μL provided Elution Buffer AM2 and incubate for 15 minutes at room temperature with end-to-end rotation. Add 50 μL provided Neutralization buffer and pipette up and down to mix. Use magnetic stand to pellet the beads and transfer the supernatant to a fresh tube. All the inputs and supernatants are then submitted to DNA purification by using the QIAquick PCR Purification Kit (Qiagen, 28106). DNA was quantified using the Quant-iT PicoGreen dsDNA Assay (Thermo Fisher Scientific, P11496). Libraries were prepared with the KAPA HyperPrep Library Kit (Roche, 07962363001) and analyzed for insert size distribution with the High Sensitivity DNA Kit (Agilent, 5067–4626) on a 2100 Bioanalyzer or the High Sensitivity D1000 ScreenTape Assay (Agilent, 5067–5584, 5067–5585, 5067–5587, 5067–5603) on a 4200 TapeStation. Libraries were quantified using the Quant-iT PicoGreen dsDNA Assay. HiSeq 2500, HiSeq 4000, or NovaSeq 6000 System (all from Illumina) was used.
GEO Accession
GSM5242673