Sequencing Data for "Unbiased RNA Degrader Identification (URID) Uncovers an LC3B-Recruiting Chimera for COL15A1 mRNA Degradation"
Description
An RNA-binding fragment (F1) with a mapped transcriptome-wide binding profile was conjugated to the LC3B ligand ispinesib to generate the degrader F1-ispinesib. Integration of RNA-seq and Chemical Cross-Linking and Isolation by Pull-down combined with NGS Sequencing (Chem-CLIP-seq) data identified collagen type XV alpha 1 chain (COL15A1) mRNA as a target selectively degraded by F1-Ispinesib. Other full plasmid sequencing files are also included here.
Files
Steps to reproduce
For RNA-seq results, MDA-MB-231 cells were seeded into 12-well plates to reach 40% confluency. After 16 h, cell culture medium was refreshed, and cells were treated with DMSO or F1-ispinesib (0.1% v/v) for 48 h. During the treatment, cell culture medium was refreshed again at 24 h and cells were re-dosed with DMSO or F1-ispinesib (0.1% v/v) for another 24 h. Total cellular RNA was harvested by using Quick-RNA Miniprep Kit (Zymo, Cat# R1054) with DNase digestion, quantified by Qubit 2.0 Fluorometer (Invitrogen), and was further assessed by Agilent 2100 Bioanalyzer RNA nanochip in terms of RNA integrity. For each biological replicate, 300 ng of extracted total RNA was depleted of ribosomal RNA using NEBNext rRNA Depletion Kit (NEB, Cat# E6310), followed by RNA-seq library preparation using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, Cat# E7760) as previously described.6 Briefly, RNA was fragmented, reverse transcribed, end prepared and ligated with adaptor sequence. After USER (Uracil-specific excision reagent) degradation of the second strand containing dUTP, cDNA was finally PCR enriched with unique index sequence. The amplified cDNA libraries were loaded into a NextSeq 500 v2.5 flow cell for sequencing (2 x 40 bp, paired-end). For each biological replicate, the obtained fasta files were aligned to human genome by STAR7 (uniquely mapped reads > 75% for each sample). The number of reads per transcript was quantified by featureCounts8, and statistical comparison of transcript abundance between samples was performed by Deseq2.9 (set cut-off as minimum read count of 10). For Chem-CLIP-seq, raw sequencing data are from a published study (DOI: 10.1021/jacs.2c01929 ). After following the same STAR alignment strategy, the number of reads per transcript was quantified by featureCounts for both input and pulldown samples and was further normalized to the sequencing depth of each replicate. For each gene that was affected by compound (F1-ispinesib) treatment, its fold of enrichment was defined as the ratio between the normalized read counts in each pulldown sample and the normalized read counts in the corresponding input sample (n = 2 biological replicates) that could be aligned to the target gene.
Institutions
- Scripps Research Institute