ribose-seq of HEK293T RNase H2 KO (T3-8) genomic DNA with RE1 fragmentation
To construct ribose-seq libraries with human cell lines, genomic DNA samples were fragmented using NEBNext dsDNA Fragmentase or a combination of restriction enzymes to generate small DNA fragments. Various sets of restriction enzymes were applied to fragment human genomic DNA samples, as indicated in table S1. The used combinations were (i) RE1: [HpyCH4V] + [Hpy116II, Eco53KI, RsaI and StuI]; (ii) RE2: [AleI, AluI and PvuII] + [DraI, HaeIII and SspI]; (iii) RE3: [CviKl-1] + [Mly, MscI and MslI].After fragmentation with restriction enzymes or NEBNext dsDNA Fragmentase, DNA was purified (QIAquick Kit). For Fragmentase-treated samples, NEBNext End Repair Module created blunt ends. Fragments underwent dA-tailing using Klenow Fragment (35 exo-), followed by adapter ligation (T4 DNA ligase, 16C/overnight) and purification. Adapter-ligated DNA was treated with 0.3 M NaOH (55C/2h) to denature strands and cleave rNMP sites, then neutralized (HCl) and purified.ssDNA fragments were circularized using Arabidopsis tRNA ligase, linear DNA removed with T5 Exonuclease, and 2-phosphates eliminated via Tpt1 treatment. Two PCR rounds (6 + 11 cycles) with Q5 polymerase added Illumina indexes, followed by size selection (250700 bp) and normalization. Libraries were sequenced on Illumina NextSeq 500/HiSeq X Ten platforms