title: GSM8768017: SplenocyteTreg_WGEM-seq_25ng_04; Mus musculus; OTHER
library_construction_protocol: Genomic DNA was extracted from flow cytometry-sorted cells using the AllPrep DNA/RNA Micro Kit. Genomic DNA was quantified with a Qubit 3.0 instrument. For WGEM-seq libraries, gDNA was first fragmented to a size of 240–290-bp using the Covaris E220 sonicator and then enzymatically converted with TET2 and APOBEC (New England Biolabs) per the manufacturer's instructions. For WGBS libraries, gDNA was bisulfite converted with the EZ DNA Methylation-Lightning Kit (Zymo Research) per the manufacturer's protocol. Additional restriction enzyme digestion step was included for RRBS and RREM-seq libraries, in which gDNA was fragmented with MspI (New England Biolabs) and then size-selected for 100- to 250-bp fragments using solid-phase reversible immobilization beads (MagBio Genomics). CG-enriched gDNA was then bisulfite converted for RRBS libraries and enzymatically converted for RREMseq libraries. Random priming, adaptor ligation, PCR product clean-up, and final library amplification were performed using the NEBNext Enzymatic Methyl-seq Kit (New England BioLabs) for WGEM-seq libraries and the Pico Methyl-Seq Library Prep Kit (Zymo Research) was used for WGBS, RRBS, and RREM-seq libraries. Libraries were run on a High Sensitivity chip using an Agilent TapeStation 4200 to assess size distribution and overall quality of the amplified library. Fluorometric quantitation and TapeStation size distribution estimates permitted equimolar pooling, and four pooled libraries per run were sequenced on an Illumina NextSeq 2000 instrument using the 200 cycles NextSeq 2000 P3 reagent kit (whole-genome libraries) or the 100 cycles NextSeq 2000 P2 reagent kit (reduced representation libraries).