title: GSM5949204: Subcutaneously vaccinated mice; Mus musculus; RNA-Seq
library_construction_protocol: Lungs were harvested from challenged animals (10 SC mice and 14 IN mice) and dissociated in Miltenyi MACS tubes (Miltenyi Inc., Germany) and digested with collagenase (1 mg/mL) and DNase (1 µg/mL) for 25 min at 37 °C. Digested lungs were resuspended in 5 mL of 40% percoll, and 3 mL of 66% percoll was underlaid (GE healthcare 17–0891–01). Samples were spun for 20 min at 2000 rpm at room temperature. Lymphocytes were then harvested from the buffy coat layer and resuspended in complete RPMI (10% FBS, 1% penicillin and streptomycin). Lymphocytes from each treatment group were pooled, resulting in two cell samples (SC and IN).  The cells were spun down (1500 rpm/5 minutes at room temperature) and stained with LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (Invitrogen) and Fc Block (BD) for 10 min at room temperature. Then the cells were stained with Bl-Eng2 tetramer (MHC class II tetramer-PE, NIH) for 1 hour at room temperature, and 30 minutes at 4°C with the following surface antibodies: CD8 PerCP-Cy5.5 (clone 53–6.7, Biolegend, cat#100734), CD44 BV650 (clone IM7, Biolegend, cat#103049), CD11b APC (clone M1/70, Biolegend, cat#101212), CD11c APC (clone N418, Biolegend, cat#117310), NK1.1 APC (clone PK136, Biolegend, cat#108710), B220 APC (clone RA3–62B, Biolegend, cat#103212), CD4 BUV737 (clone RM4–5, BD, cat#565246), and CD90.2 BV421 (clone 30-H12, Biolegend, cat105341). All panels included a dump channel to decrease background in CD4+ T cells (Dump: CD11b, CD11c, NK1.1, and B220).  The cells were fixed in 2% paraformaldehyde and sorted using the cell sorting flow cytometer FACSAria (BD). Following fluorescent labeling, cells from 10-15 animals from each vaccine (SC or IN) group were combined into one tube each for cell sorting.  Tetramer+ cells were sorted into microcentrifuge tubes containing RPMI media on a FACs Aria using a 130 micron nozzle. The sorted cells (Live, Dump-CD90.2+CD4+CD44+Tetramer+) were collected directly into 1.5 ml microtubes Sorted tetramer+ cells were counted on a Countless II cell counter with 0.4% trypan blue and concentrated to 300-400 cells/ml (total volume of 43.3 ml) and reverse transcribed. The libraries were generated with the 3' kit version 3.1 chemistry (10x Genomics).