|
| Status |
Public on Sep 07, 2023 |
| Title |
BMDM TNFa+PGE2 stimulation 2 hour RNA-seq replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
Bone marrow
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Bone marrow
strain: C57BL/6N
cell line: --
cell type: Bone marrow derived macrophages
genotype: WT
treatment: TNFa(10 ng/mL) + PGE2 (1uM)
time: 2h
|
| Treatment protocol |
BMDMs were stimulated with reagents at the following concentrations: TNFa(10 ng/mL), PGE2 (1µM), alone or in combination for 1, 2 or 4 hours respectively. KPC cells were stimulated with IL-1b (10 ng/mL) for 24 hours. Controls were left untreated.
|
| Growth protocol |
For BMDMs, bone marrrow cells were counted and seeded in IMDM supplemented with 20% FBS, 20% L929-conditioned media containing M-CSF, antibiotics (penicillin G 100 U/ml and streptomycin sulfate 100 U/ml), 2 mM L-glutamine and 5 µM 2-mercaptoethanol. Four days after culture, fresh medium was added to the cells. At day 7 after plating, cells were stimulated as described below. KPC (K8484) cell lines were previously established from tumors arising in genetically engineered mouse model carrying the G12D oncogenic mutation in the Kras gene and the missense point R720H mutation in the Tpr53 gene (KrasLSL-G12D/+;Tpr53LSL-R270H/+;Pdx1Cre/WT) and were cultured under standard conditions.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was purified using the ReliaPrep RNA Cell Miniprep System.
RNA-Seq libraries were generated using the Smart-seq2 method with minor modification. Briefly, five ng of RNA were retrotranscribed, cDNA was PCR-amplified (15 cycles) and purified with AMPure XP beads. After purification, the concentration was determined using Qubit 3.0 and size distribution was assessed using Agilent 4200 TapeStation system. Then, the tagmentation reaction was performed starting from 0.5 ng of cDNA for 30 minutes at 55°C and the enrichment PCR was carried out using 12 cycles. Libraries were then purified with AMPure XP beads, quantified using Qubit 3.0, assessed for fragment size distribution on an Agilent 4200 TapeStation system.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
Smart-seq2
|
| Data processing |
Reads were aligned using STAR aligner (v STAR_2.5.3a). Read counts matrices were computed using the featureCounts function from Rsubread package (v 2.0.1).
Assembly: mm10
Supplementary files format and content: Tab-separated counts file (gzipped format)
|
| |
|
| Submission date |
Nov 12, 2022 |
| Last update date |
Sep 07, 2023 |
| Contact name |
Renato Ostuni |
| Organization name |
San Raffaele Telethon Institute for Gene Therapy
|
| Street address |
Via Olgettina 58
|
| City |
Milano |
| ZIP/Postal code |
20132 |
| Country |
Italy |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE217842 |
IL-1b+ tumor-associated macrophages fuel pathogenic inflammation in pancreatic cancer - Bulk RNA-seq |
| GSE217847 |
IL-1b+ tumor-associated macrophages fuel pathogenic inflammation in pancreatic cancer |
|
| Relations |
| BioSample |
SAMN31700959 |
| SRA |
SRX18243135 |
