biostudies-arrayexpressNeus VisaHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16220LORAX-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.biostudies-arrayexpressLibrary Construction - 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 - Paired end-sequencing was done using the NextSeq 2000 P2 cartridge (Illumina).Sample Collection - Cells were plated and grown to ~40-50% confluency. To induce auxin-mediated DIS3 degradation, cells were treated with 500 μM Auxin for 2h, and samples were collected. LORAX-seq was performed according to published protocols (DOI: https://doi.org/10.1016/j.molcel.2024.01.019). The eluted RNA was isolated from beads using TRIZOL (Invitrogen).OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - The raw reads were pre-processed with nf-core/rnaseq v3.21.0 (https://nf-co.re/rnaseq/3.21.0/) executed with Nextflow v25.04.7. Briefly, raw FASTQ files were quality-checked with FastQC (v0.12.1), adapters trimmed with trimgalore (v0.6.10), and reads were aligned to GRCh38.p14 using STAR (v2.7.11b).Data Transformation - Alignment files were converted to bigwig format with spike-in normalization using samtools and deepTools (v3.5.5).MetabolomicsUnknownTranscriptomicsGenomicsProteomicsNextSeq 2000ChIP-seqHomo sapiensNeus VisafalseThe effect of DIS3 depletion on RNAPII backtracking in a human cell lineLORAX-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.2026-08-03T00:00:00Z2026-08-03T10:01:44.918Z2025-11-20T09:49:49.201ZE-MTAB-16220ERP185393EFO_0004170EFO_0002692EFO_0004917EFO_0005518EFO_0003816EFO_0004184