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Series GSE57138 Query DataSets for GSE57138
Status Public on Jun 15, 2014
Title Next Generation Deep Sequencing Facilitates Quantitative Analysis of microRNA affected by thapsigargin treatment
Organism Mus musculus
Experiment type Non-coding RNA profiling by high throughput sequencing
Summary Purpose: microRNA profiles were generated from NIH-3T3 cells control and thapsigargin treated, in duplicate.
The goal of this study was to compare microRNA profiles of untreated and thapsigargin treated NIH-3T3 fibroblast cells.
Methods: NIH-3T3 cells were grown to confluency and either untreated or treated with 500 nM thapsigargin in media for 24 hours. Cells were harvested with TriZol and RNA isolated according to manufacturers protocol
Analysis Outline: Short reads in fastq format were assembled using script from Illumina CASAVA 1.8.1 software pipeline.Read quality was examined using FastQC program ( Adapters were trimmed at the 3'end using Btrim prgram (PMID:21651976), only sequences equal to and longer than 18nt were retained, leading N base was trimmed at the 5' end. Unique reads were collapsed using Raw_data_parse program from miRExpress suite (PMID:19821977) (the result of this process is a file that contains unique sequences in one column and number of times this sequence was found in the library in another). They can be found in *.merge files in trimmed_reads directory. Collapsed reads were reformatted and uploaded into miRanalyzer web-based pipeline (; PMID:21515631) and matched to known mature miRNA (miRBase vesion 16), RFAM database (version 15) of known non-coding RNAs and known gene transcripts. The purpose of miRAnalyzer analysis was to only detect known miRNAs, prediction of novel miRNAs was not performed; search parameters were kept at default. MiRanalyzer output is saved in miRanalyzer folder with detailed information about mapping to known miRNA. Known miRNAs were divided into mature, maturestar (star sequences), maturestarunobs (star sequences not in miRBase) and hairpin. For each of the libraries there are files with unique and ambiguous mappings. Differentional expression analysis was based on unique alignments to known miRNAs (mature_unique.txt file in miRanalyzer folder). Mature_unique.txt has following columns: name: mature miRNA ID from miRBase; #unique reads: number of unique reads mapped; readCount: number of reads mapped; norm_expressed_all: normalized to all reads; norm_expressed_mapped: normalized to mapped reads. miRNA expression profiling was performed using edgeR bioconductor package (PMID:20478825). For differential expression analysis, used TMM normalization and analysis using common disperion (using tagwise dispersion yielded the same results). FDR was calculated according to Hochberg-Benjamini procedure (PMID:2218183). Results of differential expression analysis were saved in diff_exp folder as diff_exp.txt. diff_exp.txt contains miRNA concentrations in log scale, log2 ratio of WT to KO; p-values and FDR corrected p-values. miRNAs were sorted by p-value.
Overall design NIH-3T3 cells grown to confluency and treated with 500 nM thapsigargin in media for 24 hours
Contributor(s) Michalak M, Groenendyk J
Citation(s) 24917591, 26484121
Submission date Apr 28, 2014
Last update date May 15, 2019
Contact name Marek Michalak
Phone 780-492-2256
Organization name University of Alberta
Department Biochemistry
Lab 3-55 MSB
Street address 87 Avenue
City Edmonton
State/province Alberta
ZIP/Postal code T6H 2S7
Country Canada
Platforms (1)
GPL11002 Illumina Genome Analyzer IIx (Mus musculus)
Samples (4)
GSM1375876 control rep1
GSM1375877 control rep2
GSM1375878 thapsigargin rep1
BioProject PRJNA246123
SRA SRP041645

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Supplementary file Size Download File type/resource
GSE57138_RAW.tar 1020.0 Kb (http)(custom) TAR (of TXT)
GSE57138_diff_exp.xlsx.gz 31.3 Kb (ftp)(http) XLSX
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Processed data are available on Series record
Raw data are available in SRA
Processed data provided as supplementary file

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