PRJNA324703: RNA-Seq data for AKT, BAD, ERBB2, IGF1R, RAF1 and KRAS(G12V) overexpressed samples with twelve green fluorescent protein control samples using human mammary epithelial cells
概要: The goal was to capture the transcriptional activity due to over-expression of AKT, BAD, ERBB2, IGF1R, RAF1 and KRAS(G12V) genes .Overexpressions were validated using Western Blots. Illumina RNA-Seq technology was used to capture the downstream transcriptional activity. Reads were 101 base pairs long and single ended. An R open source package “Rsubread” was used to align and quantify the read using UCSC hg19 annotation. The integer-based gene counts were later normalized in TPM.
项目整体设计: Profiles of gene expression, downstream of AKT, BAD, ERBB2, IGF1R, RAF1 and KRAS(G12V) over-expression, were generated in cells derived from breast and used to generate a gene-expression signatures.
Human mammary epithelial cells, grown in Stemgent WIT-P culture medium at 3% CO2 and 37°C
处理方案:
Genes were introduced for over-expression by way of adenovirus. Cells were infected with MOI 500 Adenovius for 18 hours for GFP18, AKT, BAD, HER2, IGF1R and RAF1; for 30 hours for GFP30 and KRAS(G12V).
提取方案:
Cells were scraped into RNA-Later and stored on ice until processing. Then RNA was isolated from pelleted cells using a Qiagen Rneasy mini kit (with Rnase-free DNase set) and QIAshredders.
