GSM7728324: LA1_scRAGE_seq; Mus musculus; RNA-Seq
RAGE-seq libraries were prepared using a modified version of the protocol described in the original RAGE-seq study (https://www.nature.com/articles/s41467-019-11049-4). After preparation of 10x RNA-seq and CARLIN libraries, the remaining cDNA from step 2.3 of the 10x 3' v3.1 User Guide (CG000204 Rev D) was used as input into the RAGE-seq protocol. cDNA was amplified for 20 cycles and V(D)J sequences enriched with the xGen Hybridization Capture chemistry (IDT) using a custom probe set. Probes were designed against all mouse functional V, D, J and TCR/BCR constant regions obtained from the IMGT database . Following hybridization capture, libraries were further amplified for 20 cycles. Each RAGE-seq sample (1 per 10x library) was prepared for nanopore sequencing using the ligation sequencing kit for either the v10 (Sample LA1, SQK-LSK110) or v14 (Samples LA2 and LA3, SQK-LSK114) chemistry and sequenced using the corresponding FLO-MIN110 or FLO-MIN114 flow cells. Super accuracy basecalling was performed off instrument using dorado v0.2.1 on an Nvidia A40 GPU. After 7 days of infection, CARLIN mice (LA1, LA2, LA3) were euthanized, and their spleens were harvested. The spleens were mashed and filtered to get a single cell suspension. Next, Erythrocytes were depleted and the samples were enriched for CD8+ T cells. The enriched CD8+ T cells were stained and finally, live, OVA-Tetramer-BV421 and OVA-Tetramer-PE double positive CD8+ T cells were sorted.