|
| Status |
Public on Feb 13, 2014 |
| Title |
TPP_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: TNFa+PGE2+P3C
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 |
SAMN02378225 |
| SRA |
SRX365453 |
