DiMeLo-seq - CENPA: Sample 7210079.00
CENP-A DiMeLo-seq using Oxford Nanopore Technologies (ONT) long-read sequencing was performed by modifying the original DiMeLo-seq protocol and the FindingNemo genomic DNA (gDNA) extraction and ONT library preparation protocols. Briefly, 7x106 cells were resuspended in 1 ml of Dig-Wash buffer [20 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1 tablet of cOmplete, EDTAfree Protease Inhibitor Cocktail per 50 ml buffer, 0.1% w/v BSA, and 0.02% Digitonin] and incubated on ice for 5 mins to isolate nuclei. Nuclei were pelleted and resuspended in 200 µl of Tween-Wash buffer (20 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1 tablet of cOmplete, EDTA-free Protease Inhibitor Cocktail per 50 ml buffer, 0.1% w/v BSA, and 0.1% w/v Tween20) containing a primary antibody at a 1:50 dilution. Primary antibodies used in this study were mouse anti-CENP-A antibody or rabbit anti-IgG antibody. Samples were rotated overnight on a nutator at 15 rpm and 4°C to allow for antibody-binding. A mouse nanobody-Hia5 (Nb-Hia5) was prepared as a secondary antibody at 200 nM in 200 µl of Tween-Wash buffer. After washing away unbound primary antibody with Tween-Wash buffer, samples were resuspended in secondary antibody buffer and rotated for 2 hours on a nutator at 15 rpm and 4°C. The methyl donor S-Adenosyl Methionine was prepared at 800 µM in 100 µl of activation buffer (15 mM Tris, pH 8.0, 15mM NaCl, 60 mM KCl, 1 mM EDTA, pH 8.0, 0.5 mM EGTA, pH 8.0, 0.05 mM Spermidine, and 0.1% w/v BSA). After washing away unbound Nb-Hia5, samples were resuspended with SAM activation buffer and incubated at 37°C for 2 hours to activate the DNA adenine methyltransferase activity of Hia5. Following incubation, samples were collected and resuspended in 400 µl of cold PBS for gDNA extraction using the Monarch HMW DNA Extraction Kit for Cells and Blood by following the FindingNemo protocol Extracted gDNA was kept at RT for at least 48 hours to improve DNA homogeneity. For ONT library preparation, the FRA (transposase fragmentation mix) in the Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114, ONT) was adjusted by adding 8 µl for optimal DNA fragmentation, and library DNA was eluted in 100 µl of 10 mM Tris-HCl (pH 9.0) for a single library load, following the modified FindingNemo protocol. The DNA library was loaded onto a PromethION 24 R10.4.1 flow cell (FLOPRO114M, ONT) and sequenced with MinKNOW v25.03.7 (ONT). To enrich centromeric sequences during sequencing, we used the deplete mode of adaptive sampling enabled in MinKNOW to exclude sequencing reads originating from outside centromeric regions. The depletion BED file was made based on centromere annotations of the CHM13v2.0 reference genome. Libraries were retrieved and reloaded after 20-24 hours, depending on pore availability. In order to obtain sufficient coverage, two DNA libraries were prepared for some samples. Both anti-CENP-A and anti-IgG DiMeLo-seq ONT data were basecalled with dorado v0.9.6+0949eb8d. Anti-CENP-A and anti-IgG ONT data were merged in a single fastq per sample.