GSM5290398: NOMEseq_MM1S_A485_6hours_Replicate2; Homo sapiens; OTHER
MM1.S cells were trypsinised and collected by centrifugation at 500g for 5 minutes, then washed once in ice-cold PBS. Pellets were then resuspended in 1mL ice-cold Nuclei Extraction Buffer (10mM Tris pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1mM EDTA, 0.5% Igepal, plus protease inhibitors) per 5 x 106 cells and incubated on ice for 5 minutes. Nuclei were recovered by centrifugation at 500g for 5min and washed in Nuclei Wash Buffer (10mM Tris pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1mM EDTA, plus protease inhibitors). Freshly prepared nuclei (2.5 x 105 cells) were resuspended in 94.5uL 1X M.CviPI reaction buffer (NEB), then treated with 200 units of M.CviPI (NEB; 50,000 units/mL) in 5uL 10X reaction buffer, 45uL 1M sucrose and 1.5uL 32mM SAM. Nuclei were incubated for 7.5 minutes at 37°C then spiked with an additional 100 units of M.CviPI and 1.5uL SAM and incubated for an further 7.5 minutes. Reactions were immediately quenched by the addition of an equal volume (150uL) of Stop Solution (20nM Tris pH 7.9, 600mM NaCl, 1% SDS, 10mM EDTA, 400ug/mL Proteinase K) and incubated overnight at 55°C. DNA was purified using MicroChIP DiaPure columns (Diagenode) and conversion was performed using the EM-seq Conversion Module (NEB). Bisulfite conversion was performed using the EZ DNA Methylation-Gold Kit (Zymo Research) after which sequencing libraries were prepared using the Accel-NGS Methyl-Seq DNA Library Kit for Illumina (Swift Biosciences) and Methyl-Seq Set A Indexing Kit (Swift Biosciences). Indexed libraries were pooled equimolar and sequenced on a NovaSeq 6000 in a PE150/150 run using the NovaSeq 6000 (300 cycles; Illumina). An average of 460 million paired reads was generated per sample.