mRNA-seq gene expression data for seven cell lines treated with one of three CDK4/6 inhibitors at 0.3, 1.0, or 3.0 μM for 6 or 24 hours - Dataset (ID:20372)
|HMS Dataset ID:||20372|
|Dataset Title:||mRNA-seq gene expression data for seven cell lines treated with one of three CDK4/6 inhibitors at 0.3, 1.0, or 3.0 μM for 6 or 24 hours|
|Project Summary Page(s):||Publication Summary|
|Screening Lab Investigator:||Caitlin Mills|
|Screening Principal Investigator:||Peter K. Sorger|
|Assay Description:||To compare the mechanisms of action of palbociclib, ribociclib, and abemaciclib we performed transcriptional profiling (mRNA-seq) on a panel of seven breast cancer cell lines following 6 or 24 hours of exposure to 0.3, 1, or 3 μM of drug. Log2 fold change data are provided.|
1. Cells were seeded in 12-well plates, and allowed to adhere for 24 hours at which time CDK4/6 inhibitors were added.|
2. Cells were lysed in the plates after 6 or 24 hours, and RNA was extracted using Applied Biosystems MagMax 96 total RNA isolation kit (Thermo Fisher Scientific, Waltham, MA) with DNAse digestion according to the manufacturer’s protocol. RNA was checked for quantity with a NanoDrop (Thermo Fisher Scientific, Waltham, MA) and for quality using an Agilent Bioanalyzer instrument (with RIN value > 9.0).
3. Libraries were prepared using a TruSeq Stranded mRNA sample preparation kit (Illumina, San Diego, CA) from 500 ng of purified total RNA according to the manufacturer’s protocol in a reduced reaction volume. The finished cDNA libraries were assessed for quality using a Bioanalyzer and quantified with a Quant-iT dsDNA Assay kit (Thermo Fisher Scientific, Waltham, MA).
4. The uniquely indexed libraries were multiplexed based on this quantitation and the pooled sample was quantified by qPCR using the Kapa Biosystems (Wilmington, MA) library quantification kit by the Molecular Biology Core Genomics Facility at the Dana-Farber Cancer Institute and sequenced on a single Illumina NextSeq500 run with single-end 75bp reads.
5. Reads were processed to counts using the bcbio-Nextgen toolkit version 1.0.3a (https://github.com/chapmanb/bcbio-nextgen) as follows: (1) Reads were trimmed and clipped for quality control in cutadapt v1.12; (2) Read quality was checked for each sample using FastQC 0.11.5; (3) High-quality reads were then aligned into BAM files through STAR 2.5.3a using the human assembly GRCh37; (4) BAM files were imported into DEXSeq-COUNT 1.14.2 and raw counts TPM and RPKM were calculated. R package edgeR (McCarthy et al., 2012; Robinson et al., 2010) 3.18.1 (R version 3.2.1) was used for differential analysis and generate log fold change, P-value and FDR.
|HMS Dataset Type:||RNA-Seq|
|Date Publicly Available:||2020-06-24|
|Most Recent Update:||2020-06-24|