GSM5014494: PBS ctrl; Mus musculus; RNA-Seq
The second largest lung lobes were pooled from three mock or i.n. H1/ML-RR-cGAMP vaccinated mice, tissue dissociated in RPMI containing liberase and DNase I in gentleMACS C tubes using the gentleMACS dissociator and strained through a 70μm filter for sorting on a Sony SH-800 sorter using a 100μm nozzle. Sorted CD4 T cells were washed in RPMI and counted. 5000-6000 CD4 T cells per sample were used for single cell RNA sequencing (scRNA-seq) according to the 10x Genomics protocol. Single cell suspensions were partitioned into Gel Beads in emulsion (GEMs) using the 3' Chromium Next GEM single Cell v3.1 system. 10x barcoded primers were used to reverse transcribe poly-adenylated mRNA to produce barcoded, full-length cDNA. Purified DNA was amplified, fragmented, and amplified again to attach Illumina adapter sequences by the Functional Genomics Laboratory at UC Berkeley. Libraries were sequenced on an Illumina NovaSeq SP 100SR and demultiplexed.