Healthy wild type C57BL/6 female mice were sacrificed to obtain the small intestine, lungs, liver and peripheral blood (as reference). Organs were disintegrated on a Gentle MACS Octo Dissociator using the Mouse Lamina Propria Dissociation, Mouse Lung Dissociation, and Mouse Tumor Dissociation kits, respectively (all from Miltenyi Biotec), according to the manufacturer’s instructions. Crude lysates were subjected to 40%/80% Percoll gradient centrifugation (Sigma Aldrich). Then, CD8b+ CD103+ Trm cells were isolated using Miltenyi’s AutoMACS Pro magnetic sorter and Multisort MACS kits, performing two subsequent rounds of positive selection. First, cells were labeled with anti-CD8b-biotin (eBioscience) and anti-CD103-PE (BD Biosciences) antibodies. Next, CD8b+ T cells were positively selected with Anti-Biotin Multisort Microbeads (Miltenyi Biotec). Microbeads were released, the cells incubated with Anti-PE Microbeads (Miltenyi Biotec), and subjected to a second round of positive selection to retrieve the CD103+ subset. As a reference, peripheral blood-derived, non-naïve CD8b+ CD62L- T cells were also isolated from the same animals by CD8b+ T cell separation and subsequent depletion of CD62L+ cells with anti-mouse CD62L microbeads (Miltenyi Biotec). As an additional control, whole organ samples were used (n=3), which were not isolated after organ disintegration.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from MACS-separated T cells using the RNeasy Plus Micro Kit (Qiagen) following the manufacturer's recommendations. RNA yield was measured and integrity checked on an Agilent 2100 Bioanalyzer (Agilent Technologies).
Label
Cy3
Label protocol
Three hundred pg of total RNA, with an RNA integrity number >8.0 per sample was reverse transcribed, amplified in two rounds, and Cy3 labeled by the Arcturus RiboAmp HS PLUS, Cy3 Kit (Life Technologies) according to the manufacturer's instructions. Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
Amplified samples were hybridized to 4x44k mouse whole genome microarrays using the Gene Expression Hybridization Kit (Agilent)
Scan protocol
Samples were scanned on an Agilent Microarray Scanner using one color scan setting for 4x44k array slides.
Description
20
Data processing
The scanned images were analyzed with Feature Extraction Software 10.7 (Agilent). Raw data were quantile normalized and further analyzed by Partek GS (Partek). To identify T-cell transcripts, whole organs were also processed in parallel, and used to identify mRNAs enriched in respective T cell samples (ANOVA with FDR Correction, p<0.005, >2 fold change). To assess organ-specific differences in T cell gene expression profiles, these 6411 transcripts were subjected to multidimensional scaling (MDS), ANOVA or t-tests, as appropriate (with FDR Correction, p<0.05), to identify differentially expressed genes (DEGs).
