GSM9012322: PBMC, wildtype; Mus musculus; RNA-Seq
Blood from 10-week-old female Mvk+/+ and MvkV377I/del91 mice (n=3 per genotype) was pooled into EDTA-coated collection tubes (BD Vacutainer, BD Biosciences). PBMCs were isolated by centrifugation over Ficoll-Paque Plus (GE Healthcare) and erythrocytes were lysed using 0.83% NH4Cl, 0.1% KHCO3 for 5 minutes at room temperature. Cells were stained with DAPI and anti-Ter119-PerCP/Cy5.5 (BioLegend), and double negative (DAPI-Ter119-) cells were isolated by using a BD FACSAria III Cell Sorter. Single cells were encapsulated into emulsion droplets using the 10x Chromium platform (10x Genomics, California), with a targeted output of 2000 cells. scRNAseq libraries were constructed using Chromium Single Cell 3.0 v2 Reagent Kit according to the manufacturer's protocol. Amplified cDNA and final libraries were evaluated on an Agilent BioAnalyzer using a High Sensitivity DNA Kit (Agilent Technologies Inc.). Individual libraries were diluted to 4nM and pooled for sequencing, with 150 cycle run kits (26bp Read1, 8bp Index1 and 98bp Read2) on a Nextseq500 Sequencing System (Illumina, California) to 80-90% saturation level.