title: GSM7728323: LA3_scCARLIN_seq; Mus musculus; RNA-Seq
library_construction_protocol: CARLIN libraries were prepared as described in the original CARLIN study (PMID: 32413320). Briefly, 5ng of amplified cDNA from step 2.3 of the 10x 3' v3.1 User Guide (CG000204 Rev D) was PCR amplified with CARLIN-specific, biotinylated primers (15 cycles), enriched with kilobaseBINDER streptavidin beads (Invitrogen, PN 60101), and further amplified (15 cycles) prior to 0.8x SPRIselect cleanup. Samples were indexed using the 10x Genomics “TT” dual-index plate (PN 1000215). Sequencing was performed on a NovaSeq6000 using SP 500 cycle flow cells (Read1: 28 cycles; Read2: 300 cycles; Index1: 10 cycles; Index2: 10 cycles) 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.