GSM8967918: Tem.Spleen.1#1; Mus musculus; RNA-Seq
Single-cell suspensions of spleens were prepared by mechanical disruption, followed by ACK-lysing buffer. Prostate tissue was dissected from the bladder and seminal vesicles, cut into small pieces, the incubated shaking at 37°C for 30 minutes in RPMI with 1.2 mg ml−1 of Hepes, 292 µg ml−1 of L-glutamine, 1 mM MgCl2, 1 mM CaCl2, 5% FBS and 100 U ml−1 of collagenase (Worthington). Lymphocytes from the prostate were separated on a 44%/67% Percoll density gradient.For each replicate, 3 mice were pooled and then sorted for ultralow-input RNA-seq following following Immgen guidelines. A total of 5,000 live P14 T cells were sorted from the spleen and prostate at 37-38 days post infection into PBS supplemented with 5% BSA, 2% FBS and 0.4% 0.5M EDTA. A total of 1,000 P14 T cells were then resorted into 5 µl of 1x TCL buffer (Qiagen) with 1% 2-mercaptoethanol. Cell sorted from the prostate were gated on the i.v.- population. Library preparation for ultralow-input RNA-seq was carried out as described at Immgen (https://www.immgen.org/Protocols/ImmGenULI_RNAseq_methods.pdf), excerpt here: Smart-seq2 libraries were 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 18 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 Smart sequencing. Smart-seq paired-end sequencing was performed on an Illumina NextSeq500 using 2 x 25bp reads with no further trimming.