GSM8431607: W8, Rep2, bACE; Mus musculus; OTHER
To isolate gDNA, brain was thawed and digested in lysis buffer (50mM Tris, pH8.0, 100mM EDTA, 0.5% SDS) with proteinase K (Sigma-Aldrich) overnight at 55°C. Genomic DNA was isolated using Phenol:Chloroform:Isoamyl Alcohol (Fisher BP17501-400) and ethanol precipitation. DNA samples resuspended in TE were stored at 4°C. Purified mouse gDNA (200 ng) with 1 ng each of phiX, mCpG pUC19 (Zymo), and 5hmC T4 spike-in controls were sheared to approximately 350 bp using a Covaris instrument. Sheared gDNA was taken through end prep and adaptor ligation using the NEBNext Ultra II DNA Library Prep Kit (NEB). Custom-made pyrrolo-cytosine adaptors (IDT) were substituted for kit-supplied adaptors. Adapted samples were bisulfite treated using the Premium Bisulfite Kit (Diagenode) and eluted in nuclease-free water. Post bisulfite clean-up, each sample was split into two 10 µL aliquots. One aliquot was further purified with a 1.2x left-side SPRI purification (Beckman). The SPRI-purified aliquot was denatured and APOBEC3A-treated (NEB, E1725L). After A3A deamination, the sample was purified via SPRI beads (1.2x, left-sided). Quantitative PCR was used to determine the number of cycles for the indexing PCR. Indexing PCR was performed using the unique dual index primer pairs from NEBNext Multiplex Oligos for Enzymatic-Methyl Seq (NEB). After the indexing PCR, samples were purified with two 0.8x left-sided SPRI purifications. Libraries were quantified with a Qubit HS kit (Invitrogen,Q32851) and assessed for quality with an Agilent Bioanalyzer. The resulting libraries were then pooled and sequenced on an Illumina MiSeq using 150-bp read length on a nano or micro sequencing kit (Illumina). BS-Seq, bACE-Seq