The transcriptional profile of  CD8+ naive T cells was compared from F5 transgenic mice expressing normal IL-7R or a conditional IL-7R alpha that was either switched on, or switched off for different amounts of time.

Transcription profiling by array of mouse CD8+ naive T cells from mice expressing normal IL-7R or conditional IL-7R alpha switched on or off for different amounts of time

IL-7 determines T cell fitness by distinct mechanisms at different signal strengths

Affymetrix GeneChip Mouse Genome 430A 2.0 [Mouse430A_2]

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The transcriptional profile of  CD8+ naive T cells was compared from F5 transgenic mice expressing normal IL-7R or a conditional IL-7R alpha that was either switched on, or switched off for different amounts of time.


Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

Affymetrix:Protocol:Hybridization-Unknown

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-TABM-5095

Spleens were removed from F5 transgenic mice that expressed normal IL-7R or a conditional IL-7R alpha under the control of doxycycline feed. Mice were fed dox which was then removed from the diet for 0, 3 or 10 days prior to harvesting the spleens. Naive CD8+ T cells were separated using the Easysep magnet (StemCell Technologies). Cells were kept in IMDM with 0.1% BSA during this process. After separation, RNA was extracted from cells from each condition. Cells from F5 mice with normal IL-7R were also placed into culture for 24hrs in complete RPMI (RPMI 1640 supplemented with 10% heat-inactivated FCS, 50uM 2-ME  (Sigma), 200mM L-glutamine, 10mg/ml streptomycin and 10000U/ml penicillin (all from Gibco)) containing 50ng/ml IL-7 (Peprotech). Cells were cultured at a concentration of 10^6 /ml. After 24hrs, cells were harvested and RNA extracted.

P-TABM-5096

Total RNAs were prepared from each sample using the RNEasy mini kit (Qiagen) with on-column DNAse treatment. RNA quantity was measured using a nanodrop 1000 (Thermoscientific) and quality was assessed using a BioAnalyzer 2100 (Agilent). Complementary DNA was prepared by reverse transcription of the RNA sample using oligo(dT)12-18 primer (Invitrogen) and Superscript II reverse transcriptase (Invitrogen).

P-TABM-5097

Amplification of the samples was performed. Total RNA from each sample was used to generate first-strand cDNA using a T7-oligo(dT) primer. Second strand synthesis was mediated by RNAse H-DNA polymerase I. In vitro transcription was performed leading to unlabelled cRNA. A second round of cDNA synthesis was then performed followed by in vitro transcription with biotinylated nucleotides. Biotinylated cRNA was cleaned by Sample Cleanup Modules (Affymetrix) and fragmentised by metal-induced hydrolysis.

Transcription profiling by array of mouse CD8+ naive T cells from mice expressing normal IL-7R or conditional IL-7R alpha switched on or off for different amounts of time

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