|Public on Mar 10, 2015
|HER2 siRNA knockdown, replicate 3
|HER2 siRNA knockdown
|cell line: BT-474
cell line origin: female caucasian with ductal carcinoma
cell type: breast cancer cell line
transfected with: HER2-specific siRNAs
|siRNA knockdown of HER2 was achieved through the transfection of HER2-specific siRNAs (Life Technologies, Cat # 4390824, s611 and s613 ) at a final concentration of 15 nM with Lipofectamine RNAiMax at 7.5 μL/well of 6-well plate. Negative control siRNA #1 and #2 (Ambion Cat. # AM4611 and AM4613) were used as negative controls for the HER2 knockdown.
|BT474 cells were grown in Hybri-Care Medium (ATCC® 46-X™) supplemented with 10% fetal bovine serum, 100 units/ml of Penicillin, and 100 μg/ml Streptomycin at 37°C with 5% CO2.
|RNA was isolated using RNeasy® Mini Kit (Qiagen) according to the manufacturer’s protocol. An added DNase (Qiagen) treatment step was included after the first wash.
Library prepartion as per Illumina Scriptseq Complete Gold (Human/Mouse/Rat) instructions including Ribo-Zero Gold rRNA Removal Reagents.
|Illumina HiSeq 2500
|Next generation RNA sequencing of BT474 cells: After RNA was isolated from BT474 samples, we assessed the quality of RNA using BioRad Experion. RNA samples with RNA integrity number (RIN) larger than 8 (max is 10) were considered high quality and suitable for RNA-seq. Library preparation was performed using Scriptseq™ Complete Gold (Human/Mouse/Rat) (Illumina) and sequenced on Illumina Hi-Seq2500. Six samples were run on a single flow cell. We generated 100 bp paired-end strand-specific sequences, which were mapped to human genome release hg19 using TopHat with 2 mismatches allowed for full-length reads. The raw reads were mapped to human genes annotated in RefSeq database and lincRNAs annotated in Cabili et al.  using Cufflinks V2.0.2, and subsequently used for differential gene expression analysis after normalizing the values to the total mapped reads in each sample, see supporting file 14. Expression values were calculated as FPKM (fragment per kilobase of exon per million of mapped fragments) and were used to determine expression of both lincRNAs and mRNAs in HER2 knock down (KD) and negative control (NC) siRNA samples. Transcripts were considered expressed if FPKM values across either all BT474 HER2 KD samples or all NC samples were ≥ 1.0 for mRNAs and ≥ 0.25 for lincRNAs. The mean expression level was calculated, and from this statistically significant differences in expression between HER2 KD samples and NC samples was determined using a paired t-test in R. Additionally, fold changes (HER2 KD / NC) were calculated to identify differentially expressed transcripts. Transcripts were deemed differentially expressed if the fold change was ≥ 2.0 or ≤ 0.5.
Supplementary_files_format_and_content: For each sample, there are two processed data .txt files. The lincRNA files contain the XLOC number of the lincRNA described in Cabili et al., 2011 and the normalized FPKM value to the total mapped reads in each sample. The mRNA file contains the RefSeq database gene ID with corresponding normalized FPKM value.
|Aug 07, 2014
|Last update date
|May 15, 2019
|Ahmad M Khalil
|Case Western Reserve University
|Genetics and Genome Sciences
|2109 Adelbert Rd
|Integrative transcriptome-wide analyses reveal critical HER2-regulated mRNAs and lincRNAs in HER2+ breast cancer