GSM9712679: Nanopore 5mC data from empty-vector expressing 32D cells after 5 days of Aza, rep 2; Mus musculus; OTHER
32D cells expressing either the empty vector (EV) or the Idh1-R132 mutant were treated with AGI or AZA for 5 days. At the end of treatment period, 106 cells were harvested for DNA isolation. For each genotype (EV and Idh1-R132) and treatment condition (AGI and AZA), two biological replicates were collected. DNA was isolated using the Qiagen DNeasy Blood & Tissue Kit according to the manufacturer's protocol for cultured cells. DNA was eluted in 50 µL pre-warmed Buffer EB twice. DNA concentrationswas quantified using the Qubit High Sensitivity (HS) dsDNA Assay kit according to the manufacturer's instructions. A total of 1000 ng of genomic DNA per sample was sheared with Covaris g-TUBE by centrifuging at 4200 rpm for 1 min, followed by an additional 1 min at 4200 rpm after inverting the tube. Fragment size distribution was assessed using 1 µl of sheared DNA with Agilent Genomic DNA ScreenTape Assay following manufacturer's instructions. Libraries were prepared using the ONT Ligation sequencing gDNA protocol with the Multiplex Sequencing Kit V14 (Cat # SQK-MLK114.96-XL, ONT, Oxford, UK) by incorporating native barcoding and long fragment buffer. For sequencing, libraries were multiplexed equimolarly and sequenced together. Sequencing was performed on a PromethION P24 ONT device (ONT, Oxford, UK) using R10.4.1 flow cells (Cat# FLO-PRO114M, ONT, Oxford, UK), with basecalling configured to detect 5mC and 5hmC in the CpG context.