GSM6280309: LNP-mRNA vaccine, delta_8; Mus musculus; RNA-Seq
Blood was retro-orbitally collected from mice. The PBMCs and plasma were isolated from blood via SepMate-15 (StemCell Technologies). 200 µl blood was immediately diluted with 800 ul PBS with 2% FBS. The diluted blood was then added to SepMate-15 tubes with 5ml Lymphoprep (StemCell Technologies). 1200 x g centrifugation for 20 minutes was applied to isolate RBCs, PBMCs and plasma. 200ul diluted plasma was collected from the surface layer. Then the solution at the top layer containing PBMCs was poured to a new tube. PBMCs were washed once with PBS + 2% FBS before being used in downstream analysis. The separated plasma was used in ELISA and neutralization assay. PBMCs were collected for single cell profiling using a 10xGenomics platform. Cell suspension were normalized to 1000 cells/ml and loaded to achieve targeted cell recovery to 10000 cells. The samples were subjected to 14 cycles of cDNA amplification. Following this, gene expression (GEX), TCR-enriched and BCR-enriched libraries were prepared according to the manufacturer's protocol (10x Genomics). All libraries were sequenced using a NovaSeq 6000 (Illumina) with 2*150 read length.