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Sample GSM1466933 Query DataSets for GSM1466933
Status Public on Mar 10, 2015
Title HER2 siRNA knockdown, replicate 3
Sample type SRA
Source name HER2 siRNA knockdown
Organism Homo sapiens
Characteristics cell line: BT-474
cell line origin: female caucasian with ductal carcinoma
cell type: breast cancer cell line
transfected with: HER2-specific siRNAs
Treatment protocol 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.
Growth protocol 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.
Extracted molecule total RNA
Extraction protocol 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.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
Data processing 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. [4] 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.
Genome_build: hg19
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.
Submission date Aug 07, 2014
Last update date May 15, 2019
Contact name Ahmad M Khalil
Organization name Case Western Reserve University
Department Genetics and Genome Sciences
Lab BRB719
Street address 2109 Adelbert Rd
City Cleveland
State/province OH
ZIP/Postal code 44106
Country USA
Platform ID GPL16791
Series (1)
GSE60182 Integrative transcriptome-wide analyses reveal critical HER2-regulated mRNAs and lincRNAs in HER2+ breast cancer
BioSample SAMN02953992
SRA SRX671588

Supplementary file Size Download File type/resource
GSM1466933_HER2KD3_lincRNA_FPKM.txt.gz 36.0 Kb (ftp)(http) TXT
GSM1466933_HER2KD3_mRNA_FPKM.txt.gz 167.4 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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