|
Status |
Public on Sep 21, 2017 |
Title |
WT_naïve_TCRgd+_3 |
Sample type |
SRA |
|
|
Source name |
WT_naïve_TCRgd+ T_cells
|
Organism |
Mus musculus |
Characteristics |
strain: C57BL/6 genotype/variation: wildtype infection: naïve tissue: Small intestine cell type: intraepithelial lymphocytes flow cytrometry markers: CD45+EpCAM-TCRab-TCRgd+CD8a+
|
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.
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|
|
Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NextSeq 500 |
|
|
Description |
WT_nai_GD
|
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
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|
|
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 |
SAMN06651617 |
SRA |
SRX2683643 |