title: GSM6640527: N cell line rep1 H; Mus musculus; OTHER
library_construction_protocol: Mitochondrial DNA of HepG2 cells, mouse brain and mouse liver were prepared using Mitochondrial DNA Purification Kit (Abcam, ab288088). NIH/3T3 and B104-1-1 cells were both purchased from ATCC (CRL-1658; CRL-1887), and cultured in DMEM (Gibco, 11965092) with 10% FBS (Gibco, 26140079). NIH/3T3 from ECACC were purchased from Sigma (93061524-1VL), but only used in sex determination. Genomic DNA of NIH/3T3 cells, B104-1-1 cells, 4-month-old male C57BL/6J mouse whole testis, 4-month-old male C57BL/6J mouse whole brain, and 2.5-month-old female C57BL/6J mouse whole brain were all prepared using Monarch Genomic DNA Purification Kit (NEB, T3010S). All the DNA samples were harshly treated with RNase A (NEB, T3018L) again prior to libraries construction to get rid of any RNA contaminants. For mammalian gDNAs, antibody enrichment was performed prior to libraries construction: 50~100 μg RNA-free DNA was diluted by TE buffer (Invitrogen, AM9849) with 0.1 M NaOH and sonicated to 100~300 nt length. The fragmentated DNA was immunoprecipitated using 25 μg anti-6mA antibody (Sigma, ABE572 or Abcam, ab151230) by gently rotating at 4℃ overnight. Pierce Protein A Magnetic Beads (Thermo Scientific, 88845) were washed and used to pulldown antigen-antibody complex. Subsequentially, the beads were treated by proteinase K (NEB, P8111S) and the 6mA-enriched DNA were then purified with DNA Clean & Concentrator Kit-25 (Zymo research, D4033). The fragmented DNA was mixed with 15% (mass percentage) spike-in probes (Supplementary Table 1) and then spited into three in the proportion of 3:4:1, each for mutation group, demethylation control and high-fidelity control. For the demethylation control, DNA fragments was denatured and then incubated at 37 ℃ for 4 hours in a 300 μL reaction containing 50 mM HEPE 7.0, 60 mM KCl, 75 μM ammonium iron (II) sulfate hexahydrate, 2 mM L-asorbic acid, 300 μM α-ketoglutarate and 2.4 μM FTO purified protein. The demethylation reaction was then treated with proteinase K (NEB, P8111S) and cleaned up by Oligo Clean & Concentrator Kits (Zymo research, D4061). OTHER: DRM-Seq