title: GSM8896250: TC_all_LN_2, GEX; Mus musculus; OTHER
library_name: GSM8896250
library_construction_protocol: For single cell RNA seq of TCs, anatomically distinct lymph nodes were pooled from 2 w old (P14) RorcVenus-creERT2 mice with 7 biological replicates per pool of lymph nodes. Lymph nodes were minced were digested in collagenase in RPMI1640 supplemented with 5% fetal calf serum, 1% L-glutamine, 1% penicillin–streptomycin, 10 mM HEPES, 1 mg/ml collagenase A (Sigma, 11088793001) and 1U/mL DNase I (Sigma, 10104159001) for 45 min at 37°C, 250 rpm. Cells were depleted of Lineage (TCRb, TCRgd, CD19, B220, NK1.1)+ cells via staining with biotinylated antibodies followed by magnetic bead negative selection. Cells were incubated with anti-CD16/32 in sorting buffer (2% FBS in PBS) for 10 minutes at 4°C to block binding to Fc receptors. Extracellular antigens were stained for 30 minutes at 4°C in sorting buffer (2% FBS, 2mM EDTA, in PBS). Cells from distinct lymph nodes were “stained” with TotalSeqTM oligo-conjugated antibodies consisting of a pool of antibodies directed against CD45 (30-F11) and H2 MHC Class I alloantigens (clone M1/42; Biolegend). Cells were washed and resuspended in sorting buffer with SYTOX blue (Invitrogen) for exclusion of dead cells. Live, CD45+Lin(Siglec-F, TCRb, TCRgd, CD19)–RORgt(Venus)+CXCR6–MHCII+ cells were then sort purified. Cells were sorted into cRPMI, before being pelleted and resuspended in RPMI-2% FBS to a final concentration of 700–1,300 cells per μl.  Single Cell Gene Expression was performed on Chromium instrument (10X Genomics) following the user guide manual for 3′ v3.1. In brief, viability of cells was confirmed above 80% with 0.2% (w/v) Trypan Blue staining (Countess II). Cells were captured in droplets. Following reverse transcription and cell barcoding in droplets, emulsions were broken and cDNA purified using Dynabeads MyOne SILANE followed by PCR amplification per manual instruction. ~10,000 cells were targeted in total. Cells from different lymph nodes were multiplexed together on one lane of 10X Chromium using Hash Tag Oligonucleotides - HTO following previously published protocol. Final libraries were sequenced on Illumina NovaSeq S4 platform (R1 – 28 cycles, i7 – 8 cycles, R2 – 90 cycles).