GSM5526015: T8.D32.SG1; Mus musculus; RNA-Seq
To identify CD8+ T cells in the vasculature of non-lymphoid tissues (small intestine, kidney, salivary gland, fat, and liver), 3 µg of CD8α (53-6.7) conjugated to APC eFluor780 was injected by i.v. into mice three minutes prior to sacrifice, as has been previously described. Cells labeled with low to no CD8α antibody were considered to be outside of the vasculature. Single-cell suspensions of splenocytes were prepared by mechanical disaggregation followed by treatment with ACK lysis buffer. Blood samples were treated with ACK lysis buffer. Small intestine intraepithelial lymphocytes were prepared through the removal of Peyer's patches and the luminal contents. The small intestine was then cut longitudinally and into 1 cm pieces, then incubated at 37°C for 30 min in HBSS with 2.1 mg/mL sodium bicarbonate, 2.4 mg/mL HEPES, 8% bovine growth serum, and 0.154 mg/mL of dithioetheritol (EMD Millipore). The kidneys, salivary glands, fat, and liver were minced into small pieces and then incubated in RPMI with 1.2 mg/mL HEPES, 292 µ/mL L-glutamine, 1 mM MgCl2, 1 mM CaCl2, 5% fetal bovine serum, and 100 U/mL collagenase (Worthington) at 37°C for 30 min. Lymphocytes from the small intestine, kidney, salivary gland, and liver were separated on a 44%/67% Percoll density gradient. For each replicate, cells from 10-15 mice were pooled and then sorted for bulk RNA-seq. 32 days after initial infection with LCMV, 5e3 P14 cells were sorted from the spleen and blood and 5e3 IV- P14 cells were sorted from the IEL, kidney, salivary gland, fat, and liver into PBS + 5% BSA. To further improve sample purity, 1e3 cells from each sample were resorted into 1x TCL lysis buffer + 1% 2-mercaptoethanol. Samples were prepared using Immgen's standard protocol for ultra-low input RNA-seq (https://www.immgen.org/img/Protocols/ImmGenULI_RNAseq_methods.pdf). Excerpt here: 5 ul of the lysates are used for each sample for Smart-seq2 library construction, prepared as  previously described (Picelli et al., 2013; Picelli et al., 2014) with slight modifications. Briefly, total RNA was  captured and purified on RNAClean XP beads (Beckman Coulter). Polyadenylated mRNA was then selected  using an anchored oligo(dT) primer (5′–AAGCAGTGGTATCAACGCAGAGTACT30VN-3′) and converted to  cDNA via reverse transcription. First strand cDNA was subjected to limited PCR amplification followed by Tn5  transposon based fragmentation using the Nextera XT DNA Library Preparation Kit (Illumina). Samples were  then PCR amplified for 12 cycles using barcoded primers such that each sample carries a specific  combination of eight base Illumina P5 and P7 barcodes and pooled together prior to sequencing.