title: Whole-genome oxidative bisulfite sequencing and data processing of mouse intestinal stem cells with old and young mitochondria
design: Whole-genome oxidative bisulfite sequencing and data processing of mouse intestinal stem cells with old and young mitochondria
library_construction_protocol: Intestinal stem cells enriched for Old or Young mitochondria were FACS sorted. Cells form 3-4 mice of both genders(designated with M or F in sample metadata) were pooled for one biological replicate, to a total of four biological replicates. DNA was isolated using the Quick-DNA Microprep Plus Kit(D4074) according to manufacturers instructions  5-mC and 5-hmC modifications were determined using oxidative bisulfite sequencing(oxBS-Seq) with the NuGEN Ultralow Methyl-Seq with TrueMethylÂ®oxBs(M01512;Tecan). 300 ngs of genomic DNA were sonicated using a Covaris S220 sonicator(Covaris) to a mean size of 250-300bp. For quantitative assessment of oxidation the DNA was spiked with 1% of control DNA-duplexes, containing C, 5-mC and 5-hmC bases at known positions. The sonicated DNA from was split into two aliquots of 150ngs, one was oxidized, and bisulfite converted, the other was mock oxidized before bisulfite conversion. oxBS libraries required 10 PCR amplification cycles, BS libraries required 8 cycles. Library quality was assessed on an Agilent TapeStation D5000(Agilent Technologies, Santa Clara, CA).