GSM5525986: Spleen_DTE_2 [RNA-seq]; 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 CD45.1 (47-0453-82) 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. Splenocytes from 4-6 mice were pooled, and then treated with DTE, collagenase, or a cold active protease as described below. Kidneys were minced into small pieces and then incubated in either collagenase or a cold active protease. Each sample comprises pooled cells from 4-6 mice. For DTE treatment, tissues were 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. For collagenase treatment, kidneys were minced into small pieces and then both  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. For digestion with cold active protease, tissues were shaken at 4°C for 30 min in PBS with 10 mg/mL protease from Bacillus Sp (Sigma), 0.5 mM EDTA, and 125 U/mL DNase (Sigma). Cold active protease digestion was quenched with an equal volume of PBS containing 20% bovine growth serum. Lymphocytes from each digestion condition for both splenocytes and kidneys were separated using a 44%/67% Percoll density gradient. 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.