wt.th1.6h.3
Protocols: Peritoneal membrane was harvested at 3h and 6h after the induction of peritoneal inflammation. One group of mice were treated with a lyophilized cell-free supernatant prepared from Staphylococcus epidermidis (SES) via intraperitoneal injection. The second group of mice were treated with SES and CD4 + Th1 cells at the same time and administrated by intraperitonal injection. Briefly, splenic naïve CD4+ T-cells were flow sorted and place in coated plates with anti-CD3e and 5μg/ml soluble anti-CD28 antibodies. Cells were cultured for 4 days in the presence of 20ng/ml murine recombinant IL-12 (R&D systems; 419-ML). The proportion of IFNY-secreting CD4+ T-cells (Th1 cells) was determined by intracellular flow cytometry using antibodies against to CD4, IFNY, IL-17, IL-4 and IL-13. Based on this analysis, T-cells were washed in ice-cold PBS, re-suspended in a 500μl aliquot of PBS reconstituted SES. At 3 and 6 hours post-inflammatory challenge, the peritoneal membrane was harvested. Peritoneal membrane sections (80mg tissue) were dissociated in 1ml buffer RLT (QIAGEN) supplemented with β-mercaptoethanol (1:100 v:v) using a handheld electric homogeniser (Benchmark Scientific). Lysate was diluted 1:3 in distilled water and digested in 0.2mg/ml proteinase-K (Invitrogen; 25530049) for 10 minutes at 55oC. Lysate was cleared and RNA precipitated in 70% ethanol. Total RNA was extracted using the RNeasy Mini kit (QIAGEN) in accordance with the manufacturer's instructions. 2-4ug of mRNA was processed to generate the libraries. Cytoplasmic, mitochondrial, and ribosomal RNA was depleted using the Ribominus transcriptome isolation kit (Ambion; K155001). Libraries were prepared using the RNA-seq kit v2 (Life technologies; 4475936)
ENA first public
2021-05-01
ENA last update
2021-02-09
External Id
SAMEA8006889
INSDC center alias
CARDIFF UNIVERSITY
INSDC center name
CARDIFF UNIVERSITY
INSDC first public
2021-05-01T00:24:01Z
INSDC last update
2021-02-09T11:40:00Z
INSDC status
public
Submitter Id
E-MTAB-10087:wt.th1.6h.3
age
10 to 12
broker name
ArrayExpress
common name
house mouse
developmental stage
adult
genotype
wild type genotype
organism part
peritoneum
sample name
E-MTAB-10087:wt.th1.6h.3
sex
male
strain
C57BL/6