|
| Status |
Public on Sep 21, 2017 |
| Title |
WT_naïve_intestinal_epithelial_cells_1 |
| Sample type |
SRA |
| |
|
| Source name |
WT_naïve_intestinal_epithelial_cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
genotype/variation: wildtype
infection: naïve
tissue: Small intestine
cell type: intestinal epithelial cells
flow cytrometry markers: CD45-EpCAM+
|
| Treatment protocol |
Mice were orally inoculated with 10^8 CFU of S. enterica serovar Typhimurium (SL1344) . Gene expression analysis was performed 18 hours after inoculation as indicated. Briefly, a single aliquot of Salmonella was grown in 3 ml of LB overnight at 37ºC with agitation, and then the bacteria were sub-cultured (1:30) into 3 ml of LB for 3.5h at 37ºC with agitation. The bacteria were next diluted to final concentration in 1 ml of DPBS. Bacteria were inoculated by gavage into recipient mice in a total volume of 100µl.
|
| Growth protocol |
N/A
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Intraepithelial lymphocytes were isolated as previously described(Mucida et al., 2007). Briefly, small and large intestines were removed and placed in chilled HBSS media (Gibco) containing 2% FCS. The intestines were carefully cleaned from the mesentery and flushed of fecal content. Intestines were opened longitudinally and then cut into 1 cm pieces. The intestinal tissue was transferred to a 50 ml Falcon tubes containing 25 ml of cold HBSS complemented with 2% FCS and 5 mM EDTA and shaken (2x) at 230 rpm for 20 min at 37°C. The tissue suspension was passed through a stainless steel sieve into 50-ml conical tubes and the cells were pelleted by centrifugation at 1200 rpm for 10 min at 4°C. The cell pellet was resuspended in complete HBSS, layered over a discontinuous 40/70% Percoll gradient, and centrifuged at 2000 rpm for 30 min. Cells from the 40/70% interface were collected, washed and resuspended in complete RPMI media. These purified cells constituted the intraepithelial lymphocyte (IEL) population. Cells were sorted using a FACS ARIAII with markers as shown above. Total RNA was extracted using the RNAeasy kit from Qiagen.
For total RNA from TCRgd IELs, RNA libraries were prepared using the SMART-Seq™ v4 Ultra™ Low Input RNA kit (ClonTech Labs) . For total RNA from intestinal epithelial cells, RNA libraries were prepared using TruSeq® Stranded mRNA Sample Preparation. All samples were sequenced using 75 base pair single end reading on a NextSeq 500 instrument (Illumina) at High Output setting.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
WT_nai_IEC
|
| Data processing |
reads were aligned to Mouse Ensembl genes using the STAR version 2.3.0 software
Differential expression was determined by use of the Cufflinks software with default settings
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text file includes RPKM
|
| |
|
| Submission date |
Mar 29, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Daniel Mucida |
| E-mail(s) |
mucida@rockefeller.edu
|
| Organization name |
The Rockefeller University
|
| Lab |
Laboratory of Mucosal Immunology
|
| Street address |
1230 York Avenue
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE97184 |
Intestinal epithelial and intraepithelial T cell crosstalk mediates a dynamic response to infection |
|
| Relations |
| BioSample |
SAMN06651609 |
| SRA |
SRX2683651 |
