Fiber-seq of SKNMC
Chromatin accessibility was profiled using the Fiberseq workflow to methylate accessible endogenous adenines (m6A) with Hia5 methyltransferase in isolated nuclei from leukemia cell lines. Briefly, cells were pelleted by centrifugation at 350 g for 5 minutes and resuspended in 2X lysis buffer to isolate nuclei. Nuclei pellets were incubated with 200 U/L Hia5 methyltransferase and S-adenylmethionine (SAM) cofactor for 10 minutes at 25C. High-molecular-weight (HMW) DNA was extracted using the MagAttract HMW DNA Kit (Qiagen), yielding genomic DNA fragments of 100-200 kb. HMW DNA was then fragmented to ~18 kb with Megaruptor 3. Libraries prepared using the PacBio SMRTbell prep kit were sequenced on PacBio's Revio platform to generate circular consensus sequencing (CCS) reads capturing both exogenous m6A (accessibility) and endogenous 5mC (methylation) modifications. CCS reads were aligned to GRCh38, and m6A-based accessibility peaks were called and stored in BAM format. Reads were phased into haplotypes using the Fiberseq/FIRE workflow pipeline based on variant calling.