The effect of DIS3 depletion on RNAPII backtracking in a human cell line

LORAX-seq (Yang et al, DOI:https://doi.org/10.1016/j.molcel.2024.01.019) was used to identify the backtracked RNA in DIS3-depleted HCT116 DIS3-AID cells. Briefly, LORAX-seq was performed according to Yang et al. (2024) with following modifications. The Pol II complexes were immunoprecipitated with α‐Rpb1 CTD (ab5408, Abcam), followed by capture using a mixture of Dynabeads Protein-A and G. The magnetic beads were treated with recombinant Human TCEA1 protein (ab204205, Abcam) as per Yang et al. (2024), followed by isolation of eluted RNA using TRIZOL (Invitrogen). RNA was prepared for sequencing using the NEBNext Low-bias Small RNA library prep kit (NEB). The eluted RNA was mixed with QIAseq miRNA Library QC Spike-in (331535, Qiagen) as per manufacturer’s instructions. Sequencing libraries were visualized using an Agilent 2100 Tapestation and sequencing was done using the NextSeq 1000/2000 P2 cartridge (Illumina). We identified longer backtracked RNAs in DIS3-depleted cells than in control cells.