Most human DNA replication initiation is dispersed throughout the genome with only a minority within previously identified initiation zones

File names indicate:

The cell lines used (HeLa-S3 denoted as HeLa; hTert-RPE1 denoted as RPE1) as the first four characters of the file name;

TE in the file name indicates that the sample was subjected to nCATS target enrichment sequencing (all other files are from genome-wide sequencing);

BAM files include both alignment (to the GRCh38 human genome assembly) and BrdU modification probabilities in the mod.bam format;

BED files list the location of left/right replication forks; initiation sites; termination sites as indicated.

Experimental protocol:

HeLa-S3 (adherent) and hTERT-RPE1 were maintained in DMEM Glutamax (HeLa-S3) or DMEM/F12 Glutamax (hTERT-RPE1, both Gibco), with the addition of 10% foetal bovine serum (Sigma) and 1% penicillin/streptomycin (Gibco). Cells were maintained at 70% confluency in 5% CO2 at 37 °C.

Cells were treated with sequential addition of 0.5 µM BrdU every 2.5 minutes until 12 µM, followed by incubated for a further 1 hr at 12 µM BrdU

High molecular weight genomic DNA extraction protocol

For the target enrichment (TE; via nCATS) samples genomic DNA was prepared for sequencing with this protocol: dx.doi.org/10.17504/protocols.io.bmi5k4g6

Libraries were prepared with ONT Ligation Sequencing Kit (SQK-LSK109 or SQK-ULK001) 

Data processing steps:

Basecalling was perform with guppy v6.1.5

Aligned reads to hg38 reference using minimap2 v2.17

Detected BrdU in reads using DNAscent v2.0.2

BrdU probabilities converted to mod.bam format with detect_to_modBAM script

BrdU gradients, replication fork directions, replication initiation and replication termination sites identified with a custom Rscript