GSM8124640: 8wk-PBS-Only-Mouse-1; Mus musculus; RNA-Seq
8 weeks old or 90 weeks old C57BL/6J mice were injected on the right back with PBS or were immunized with 200μg/mouse endotoxin-free OVA, in combination with 10μg/mouse LPS plus 65μg/mouse PGPC all emulsified in incomplete Freund's adjuvant. Immunized mice received two boost injections with the same dose of OVA+LPS+PGPC on the left back. 7 days post the last boost injection, skin draining lymph nodes from immunized mice were collected and dissociated into a single cell suspension. T cells were enriched using anti-CD3e beads (Miltenyi) following the manufacturer's protocol. Enriched T cells were filtered onto a 70um then 40um filters (Miltenyi). T cell purity was assessed by flow cytometry and was above 90% for all samples. T cells were filtered onto 30um filters before counting and were resuspended in the NOVAseq 10x master mix at a concentration 12,000 cells in 43.1 ul per sample volume. Library was performed according to the manufacturer's instructions (single cell 5' v2, 10x Genomics). Approximately 20,000 single cells per sample were loaded into each channel of a Chromium single-cell 5′ chip (v2, 10x Genomics) at the Genomics Platform of the Broad Institute. Single cells were partitioned into droplets with gel beads in the Chromium Controller. After emulsions were formed, barcoded reverse transcription of RNA took place. This was followed by cDNA amplification, amplification of full-length V(D)J segments. Fragmentation, adaptor ligation, and sample index attachment were performed on full-length cDNA and V(D)J segments to create 5' gene expression and TCR libraries, respectively. Libraries from 30 10x channels were pooled together, with the TCR libraries being pooled at 25% molarity. The libraries were sequenced across 6 lanes of an Illumina NovaSeqX flow cell (10B reads, up to 300 cycles), with paired end reads as follows: read 1, 26 cycles; read 2, 90 cycles; index 1, 10 cycles; index 2, 10 cycles.