GSM6807136: Memory CD4 T cell TCR panel; Mus musculus; RNA-Seq
Spleens were processed into a single cell suspicion using a 40mm filter, resuspended in PBS-2%FCS, 2mM EDTA (MACS buffer) and the StemCell EasySep CD4 isolation kit (19852) used to isolate CD4+ cells by negative selection. Lungs were snipped and digested with Dispase II (3.2mg/ml), Collagenase P (0.4mg/ml) and DNase I (0.2mg/ml) for 25minutes in a shaking 37°C incubator. Digested tissue was processed into a single cell suspension by passing through a 40mm filter. Cells were resuspended in MACS buffer and CD4+ cells isolated with StemCell EasySep CD4 isolation kit. Isolated CD4 cells were stimulated for 4 hours with bone marrow dendritic cells incubated the night before with sonicated influenza A virus antigen. Cells were then stained with cell surface antibodies: anti-CD4 APC-Alexa647, anti-CD44-PerCP-Cy5.5, anti-MHCII-e450, anti-B220-e450, CD8-e450, F4/80-e450 and CD45 Sample Tags (BD Bioscience 1-8) to enable multiplexing and AbSeq antibodies to ICOS (AMM2072) and PD1 (AMM2138). Cells were FACS sorted on an ARIA IIU isolating IAV specific EYFP+ CD4+ CD44hi cells from the spleen (5000 cells/mouse) and lung (1000 cells/mouse) and naïve CD4+EYFPnegative CD44lo cells from the spleen (5000 cells/mouse). Cells were combined to generate one sample. Sorted cells were combined and resuspended in BD Rhapsody sample buffer and loaded onto a scRNAseq BD Rhapsody cartridge using the BD Rhapsody reagent kit (633731) according to the manufacturer's protocol. Libraries were prepared as per the Mouse VDJ, Sample Tag, and BD AbSeq library preparation protocol (23-22688-00). In brief, single cell cDNA was prepared using BD Rhapsody kit (633773) and template switching performed using the template switch oligo: 5' T TTT TTT TTT TTT TTT TTT TTT TTT rG rG rG 3 (IDT) followed by Klenow extension (NEB: M0212L), exonuclease treatment and then library generation with the BD Rhapsody Immune Response Targeted Panel (mouse) supplemented with an extra 10 genes. At the same time, libraries were prepared for the sample Tags, Abseq antibodies. PCR products were purified by double-sided size selection and index PCR performed for each library.