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Sample GSM1242512 Query DataSets for GSM1242512
Status Public on Feb 13, 2014
Title M2_72h_rep3_miRNAseq
Sample type SRA
 
Source name peripheral blood
Organism Homo sapiens
Characteristics cell type: monocyte derived macrophages
initial differentiation: GM-CSF
activation stimuli: IL4
time: 72h
donor: Donor 12
date: Date 5
Treatment protocol To further polarize macrophages into different subtypes the following stimuli were used: M1 (3days 500IU/ml rhGM-CSF + 200IU/ml rhIFNy); M2 (3days 500IU/ml rhGM-CSF + 1000IU/ml rhIL-4); TPP (3days 500IU/ml rhGM-CSF + rhTNFα (800 IU/ml), Pam3CSK4 (P3C, 1 µg/ml), prostaglandine E2 (PGE2, 1 µg/ml).
Growth protocol For differentiation primary human monocytes into M0 macrophages, cells were cultivated for 3 days in RPMI1640 medium + 10%FCS + Pen/Strep + 500IU/ml rhGM-CSF
Extracted molecule total RNA
Extraction protocol 5x106 -2x107 MΦ were harvested and total RNA including small RNAs was isolated.
Small RNA libraries were generated from 1 μg total RNA with the TruSeq Small RNA Sample Preparation Kit (Illumina). After successful ligation of 3’ and 5’ adapters to RNA molecules, RNA was reverse-transcribed using SuperScript II reverse transcriptase (Invitrogen). cDNA was amplified by 11 PCR cycles with high-fidelity Phusion Polymerase (Finnzymes). cDNA with the size of miRNAs plus ligated adapters was purified on a pre-cast 6% Tris/Borate/EDTA polyacrylamide gel electrophoresis gel (Invitrogen).
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection size fractionation
Instrument model Illumina HiScanSQ
 
Data processing Basecalls were performed using CASAVA software version 1.8
Sequencing reads were retrieved as FASTQ files and then demultiplexed
Adapter sequences were trimmed from each read using Flicker 3.0 (Illumina)
Trimmed reads were mapped to hairpin and mature human miRNAs deposited in miRBase version 19 using the short read aligner Bowtie 0.12.9 (Langmead et al., 2009) with no mismatches allowed
The number of reads mapping to a specific miRNA sequence were counted within Partek Genomics Suite version 6.6 (PGS)
The dataset was normalized (NORM) by using the statistical software R package DESeq (Anders and Huber, 2010) and miRNAs having less than one normalized read count in all unpolarized macrophage (M0) samples were excluded
The read counts were transformed (TRANSF) into log2 counts per million (cpm) and were divided by the corresponding library size (in millions) by using the R package limma (Smyth, 2005)
The R package sva (Johnson et al., 2007) was used to perform a batch removal (BR) for the random factors date and donor. Then miRNAs having less than one transformed read count in all samples of the same condition were excluded
Differentially expressed miRNAs between macrophages polarized with IFNγ (M1), IL-4 (M2) or with the combination of TNFα, PGE2 and P3C (TPP) were determined against M0 by using the R package limma (Smyth, 2005) with a p-value of 0.05 as well as an absolute fold change of 2 as cutoffs
Finally, for each condition a set of uniquely differentially expressed miRNAs was determined, which was then sorted by the transformed expression values.
Genome_build: miRBase version 19
 
Submission date Sep 30, 2013
Last update date May 15, 2019
Contact name Joachim Schultze
E-mail(s) j.schultze@uni-bonn.de
Organization name LIMES (Life and Medical Sciences Center Genomics and Immunoregulation)
Department Genomics and Immunoregulation
Street address Carl-Troll-Strasse 31
City Bonn
State/province NRW
ZIP/Postal code 53115
Country Germany
 
Platform ID GPL15456
Series (2)
GSE47189 Transcriptome-based network analysis reveals a spectrum model of human macrophage activation
GSE51307 Transcriptome-based network analysis reveals a spectrum model of human macrophage activation [miRNA-seq]
Relations
BioSample SAMN02378223
SRA SRX365452

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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