Library quantitation kit

WGBS was performed following the PBAT strategy [29 (link)] with recent improvements using a highly efficient single-stranded DNA ligation technique [28 (link)]. The molar concentration of the sequencing library was determined using the library quantitation kit from Takara Bio Inc. (Shiga, Japan). Small-scale sequencing was performed using Illumina MiSeq with the MiSeq v3 Reagent Kit in the paired-end mode for 2 × 75 cycles (for bacterial and budding yeast cells) or with the MiSeq v2 Reagent Kit nano on the paired-end mode for 2 × 150 cycles (for lambda DNA). For large-scale sequencing of mammalian cells, paired-end sequencing for 2 × 150 cycles using HiSeq X Ten was performed by Macrogen Japan Corp. (Tokyo, Japan). The reads were mapped to a reference genome (J02459 for lambda phage, NC_000913 for bacterial cells, sacCer3 for yeast cells, and hg19 for human cells) with BMap (https://github.com/FumihitoMiura/Project-2). The mapped alignments were summarized using in-house developed pipelines (https://github.com/FumihitoMiura/Project-2).

If required, DNA fragments (100–500 bp) were prepared using Focused-ultrasonicator S220 (Covaris, Woburn, MA) with microTUBE AFA Fiber Pre-Slit Snap-Cap. The sequencing library was prepared using the ThruPLEX DNA-Seq Kit (Takara Bio Inc., Kusatsu Japan). The library yield was measured using the Library quantitation kit (Takara Bio Inc.). Small-scale sequencing was performed using the Illumina MiSeq with MiSeq Reagent Kit v3 (150 cycles) in the paired-end mode of 2 × 75 cycles. Large-scale sequencing was performed using the HiSeq X Ten by Macrogen Japan Corp. (Tokyo, Japan) in the paired-end mode of 2 × 150 cycles.