|
| Status |
Public on May 15, 2020 |
| Title |
poly(A) mRNA MDSC_p50ko_IFNg R2 |
| Sample type |
SRA |
| |
|
| Source name |
Monocytic Myeloid Derived Suppressor Cells (M-MDSC) replicate 2
|
| Organism |
Mus musculus |
| Characteristics |
strain: C56Bl6/J
genotype: p50ko
cell type: M-MDSC (28 post injection)
treatment: Interferon-treated
|
| Treatment protocol |
5 million M-MDSC were plated in RPMI1640 containing 10% FBS and left untreated or stimulated with 200U/ml interferon gamma for 4 hours.
|
| Growth protocol |
CD11b+Ly6C+ Monocytic Myeloid Derived Suppressor Cells (M-MDSC) were obtained by serial selection using magnetic separation from tumor-bearing C56Bl6/J mice, 28 days after injection of murine fibrosarcoma (MN/MCA1). In details, myeloid suppressor populations were first enriched by serial negative selections with CD19 and CD11c microbeads, and then positively selected with Ly6G microbeads. Remaining cells were positively selected with CD11b+ microbeads. The purity of the cell populations evaluated by flow cytometry exceeded 90%.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
The poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads. Following purification, the mRNA was fragmented into small fragments using divalent cations and elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. cDNA fragments were end-repaired and a single ‘A’ base was added before adapter ligation. The products were then purified and enriched by PCR to create the final cDNA library.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Reads were adapter-trimmed according to the Illumina pipeline (Bcl2fastq)
Reads were mapped to the mouse mm9 genome and transcripts using TopHat 1.3.1 (PMID: 19289445), allowing up to two mismatches and with a mean distance between pairs (-r) of 150bp
Transcripts abundance were quantified using HTSeq (PMID: 25260700)
Differentially expressed genes were called using edgeR (PMID: 19910308)
Genome_build: mm9 (NCBI Build 37), transcripts from Mus Musculus UCSC iGenome
Supplementary_files_format_and_content: Counts per million (CPM) mapped reads obtained using edgeR
|
| |
|
| Submission date |
Jul 15, 2016 |
| Last update date |
May 15, 2020 |
| Contact name |
Viviana Piccolo |
| E-mail(s) |
[email protected]
|
| Organization name |
European Institute of Oncology (IEO)
|
| Department |
Department of Experimental Oncology
|
| Lab |
Gioacchino Natoli Lab
|
| Street address |
Via Adamello 16
|
| City |
Milano |
| State/province |
Milano |
| ZIP/Postal code |
20139 |
| Country |
Italy |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE84468 |
Tumor-derived prostaglandin E2 promotes p50 NF-κB-dependent differentiation of monocytic MDSC. |
|
| Relations |
| BioSample |
SAMN05408028 |
| SRA |
SRX1951576 |
