GSM6543233: PS19, RNA; Mus musculus; RNA-Seq
Female WT, 5XFAD, PS19, and PS-5X mice were sacrificed at 9 months of age by carbon dioxide asphyxiation and spleen, deep cervical lymph nodes, and blood were gently removed prior to intracardiac perfusion with 0.01M phosphate buffered saline (PBS). Half-brains were immediately placed in complete RPMI medium, cut into fine pieces and placed in dissociation tubes along with Collagenase IV+ DNase1 in complete RPMI. Tissue was then placed into a gentleMACS Dissociator for 30 mins on the ABDK setting at 37°C and then filtered through 70μm strainers. Further removal of myelin and debris was conducted using 23% Percoll density gradient centrifugation. For sorting, cells were then stained with the following antibodies: APC-CD8, APC-Cy7-CD45, FITC-CD4, PE-TCRg/d, BV421-CD11b, and PI (all with 1:200 dilution). CD4+, CD8+, and γδ+ T cells were then sorted on a BD FACSAria II and cells from multiple mice were pooled for each genotype into 2 samples each for WT and 5XFAD and 1 sample each of PS19 and PS-5X: n=10 WT#1, n=8 WT#2, n=4 5XFAD#1, n=3 5XFAD#2, n=4 PS19, n=4 PS-5X. Single cell 5' gene expression libraries and single cell V(D)J enriched libraries were prepared according to the 10x Genomics Chromium Single Cell 5' Reagent Kits User Guide v2 (Dual Index) (CG000331 Rev C). The 10x Genomics workflow was followed according to the manufacturer protocol and libraries were pooled at equimolar concentrations for sequencing on an Illumina NovaSeq 6000, targeting 50,000 reads per cell for gene expression and 5,000 reads per cell for TCR expression.