title: GSM4875572: mESCs, BisMapR_Rep2; Mus musculus; OTHER
library_construction_protocol: R-loops were collected from supernatant after treatment with MapR protocol (Yan et al. Cell Reports 2019). Total RNA was collected using Trizol (Invitrogen) and digested with Turbo Dnase (Ambion). rRNA was selectively depleted using FastSelect (Qiagen). rRNA-depleted RNA was converted to cDNA using the Ultra II Directional RNA kit for Illumina (NEB #E7760). BisMapR converted R-loops was converted to dsDNA using the Second Synthesis buffer and enzyme mix from Ultra II Directional RNA kit for Illumina (NEB #E7760). DNA samples were end-repaired using End-Repair Mix (Enzymatics), A-tailed using Klenow exonuclease minus (Enzymatics), purified with MinElute columns (Qiagen), and ligated to Illumina adapters (NEB E7600) with T4 DNA ligase (Enzymatics).  Size selection for fragments >150 bp was performed with AMPure XP (Beckman Coulter).  Libraries were PCR amplified with dual index barcode primers for Illumina sequencing (NEB E7600) using Q5 DNA polymerase (NEB M0491) and purified with MinElute.