title: EM-seq of wild type Jurkat cells
design: We generated whole genome enzymatic methyl sequencing (WGEMS) libraries for 4 samples corresponding to two technical replicates of wild-type (untreated) Jurkat cells and Jurkat cells treated with RENDER-CRISPRoff targeting CD55. Genomic DNA was isolated using the PureLink Genomic DNA Mini Kit (Invitrogen), 200 ng of DNA was diluted with TE buffer to 108 l. Control DNA Unmethylated Lambda (NEB #E7122AVIAL) and Control DNA CpG methylated pUC19 (NEB #E7123AVIAL) were diluted 1:50 with TE buffer and 1 l was added to the genomic DNA (final volume 110 l). DNA samples were sheared using a Covaris S220 (peak incident power: 175 W, duty factor: 10%, cycles per burst: 200, treatment time: 180 s). 50 l of sheared DNA was used for enzymatic methyl conversion following the NEBNext Enzymatic Methyl-seq Kit (NEB #E7120S/L) for standard libraries using the formamide denaturation protocol. 5 ng of deaminated DNA was PCR-amplified for 6 cycles, followed by PCR-clean up and eluted in TE buffer according to protocol. Amplified libraries were quantified on an Agilent 2100 TapeStation system (Agilent) and equally pooled to a final concentration of 2.475 ng/l in 50 l (approximately 10 nM at 375 bp). The prepared libraries were sequenced paired-end 150 bp reads on an Illumina NovaSeq X 1.5B 2 Lanes (1.5 billion clusters). We obtained between 338.2M to 439.6M paired-end reads per library across all 4 libraries.