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Sample GSM4306461

Query DataSets for GSM4306461

Status

Public on Mar 10, 2020

Title

ActD time-course 0h rep3

Sample type

SRA

Source name

MCF7 cell line

Organism

Homo sapiens

Characteristics

protocol: MARS-seq (applied to bulk cells)
cell line: MCF7

Treatment protocol

CPT 6uM, 24 hours

Growth protocol

cells were grown in DMEM media (Gibco) supplemented with 10% bovine serum and 1% penicillin/streptomycin (Gibco) in 5%CO2-buffered incubators at 37 degrees.

Extracted molecule

total RNA

Extraction protocol

TRIZOL and Direct-zol RNA MiniPrep kit (Zymo research). Followed MARS-seq protocol: https://www.ncbi.nlm.nih.gov/pubmed/31101904

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

total RNA, poly(A) enriched

Data processing

MARS-seq Raw data was processed using UTAP (Kohen et al. 2019) with default parameters.
Corrected counts of control samples were normalized by Renilla mRNA spike, which was counted using Bowtie2 (Langmead et al., 2009).
Corrected counts of CPT-treated samples were normalized by mouse PolyA+ enriched RNA, which was mapped to mouse genome using STAR (Dobin et al. 2013) not allowing mismatches. Percent of uniquely mapped reads per sample was used as normalization factor.
Biological replicates were averaged and means were used for fitting a nonlinear least-squares model assuming first-order decay kinetics, while corrected counts at t=0 were used as C0 and inverse of standard errors of the corrected count means were used as weights for fitting. Half lives of all genes were then calculated. Genes with mean corrected read count < 5 at t=0 were removed from subsequent analysis. Negative half life values and half lives > 24h were set to 24h.
RNA-seq analysis: Since mouse PolyA+ enriched RNA was used for normalization, reads were first mapped to mouse genome (mm9) using STAR and unmapped reads from this step were remapped to the human genome (hg19 assembly). For normalization, reads were mapped separately to the mouse genome not allowing mismatches and percent of uniquely mapped reads per sample was used as normalization factor. Gene expression levels were quantified using RSEM (Li and Dewey, 2011) and Bowtie2. GENCODE v26 annotations were used for this and all subsequent analysis, with all histone genes removed from annotation. TPM values were normalized by mouse normalization factors and filtered by a minimal value of 1 TPM. Means of the two replicates were used for subsequent analysis.
Genome_build: hg19; GENCODE v26 annotation was used for this analysis.
Supplementary_files_format_and_content: An Excel spreadsheet including calculated half-life values measured in hours (using MARS-seq data) and abundance measurements estimating transcription levels gene-level TPM values obtained by RSEM quantification of the data for each gene (using RNA-seq data).

Submission date

Feb 11, 2020

Last update date

Mar 10, 2020

Contact name

Binyamin Zuckerman

Organization name

Gladstone Institute

Street address

1650 Owens Street

City

San Francisco