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Re-analysis of PRO-cap data from HCT116 cells (GSE219977)

ABSTRACT: This submission provides processed PRO-cap coverage tracks (bigWig files) from the re-analysis of public data (GSE219977, samples GSM6783554 and GSM6783555) from Homo sapiens HCT116 genetically modified (insertion) using CRISPR inserting O. sativa LOC4335696, (insertion) using CRISPR targeting H. sapiens SUPT16H. Data were aligned to the hg38 reference genome (GENCODE release 33; https://www.gencodegenes.org/human/release_33.html) and normalized using bamCoverage (PMID: 27079975). These processed files are intended for visualization and downstream analysis of nascent transcription.

ORGANISM(S): Homo sapiens

PROVIDER: GSE300325 | GEO | 2026/03/09

REPOSITORIES: GEO

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Project description:Purpose: To identify regulatory proteins that are potential drivers of a coordinated breast cancer metastasis gene expression signatures. Methods: Knockdown of target genes in breast cancer cell lines was achieved using scramble and/or gene-specific siRNA (ON-TARGET SMARTpool, Thermo Scientific) and Lipofectamine RNAiMAX. 48h post transfection, total RNA was isolated from cell lines using the RNeasy Plus mini prep kit (Qiagen). Nucleic acid quality was determined with the Agilent 2100 Bioanalyzer. RNA Sequencing was also performed at the New York Genome Center (Manhattan, NY, USA) using a HiSeq 2500 Ultra-High-Throughput Sequencing System (Illumina, San Diego, CA, USA). Results: Raw reads in the fastq format were aligned to Human Genome HG19 using the RNA-seq STAR aligner version 2.4.0d (http://www.ncbi.nlm.nih.gov/pubmed/23104886, http://www.ncbi.nlm.nih.gov/pubmed/26334920) as recommended by user manual downloaded along with the software. STAR aligner was chosen for mapping accuracy and speed (http://www.nature.com/nmeth/journal/v10/n12/full/nmeth.2722.html). Mapped reads for each sample were counted for each gene in annotation files in GTF format (gencode.v19.annotation.gtf available for download from GENECODE website (http://www.gencodegenes.org/releases/19.html)) using the FeatureCounts read summarization program (http://www.ncbi.nlm.nih.gov/pubmed/?term=24227677) following the user guide (http://bioinf.wehi.edu.au/subread-package/SubreadUsersGuide.pdf). Individual count files were merged to generate the raw-counts matrix by an in-house R script, normalized to account for differences in library size and the variance was stabilized by fitting the dispersion to a negative-binomial distribution as implemented in the DESeq R package (http://bioconductor.org/packages/release/bioc/html/DESeq.html)(Anders and Huber, 2010). Conclusions: Our data suggest that targeting keystone proteins in the breast cancer metastasis transcriptome can effectively collapse transcriptional hierarchies necessary for metastasis formation, thus representing a formidable cancer intervention strategy.

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Re-analysis of PRO-cap data from HCT116 cells (GSE219977)

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