GSM8153454: Total LSK/Tom+ LSK cells, HTO; Mus musculus; OTHER
Tom+ Lin- Sca-1+ c-Kit+ (LSK) cells from Kras reporter mice (n=2) or total LSK cells from WT reporter mice (n=2) were sorted. Single cells were encapsulated into emulsion droplets using Chromium Controller (10x Genomics) and subsequent scRNA-seq and Cell Surface Protein libraries were constructed using Chromium Single Cell 3′ v3.1 Reagent Kit according to the manufacturer's protocol. Briefly, cell populations were hashtagged using BioLegend Type B oligo-tagged antibodies and counted using a BioRad TC20 Automated Cell Counter. Cells were then pooled and placed in each channel of a Next GEM Chip, loaded onto the Chromium instrument and an emulsion was created. Reverse transcription and library preparation were performed on a C1000 Touch Thermal cycler with 96-Deep Well Reaction Module (Bio-Rad). cDNA was amplified 11 cycles and evaluated on a Agilent BioAnalyzer 2100 using a High Sensitivity DNA Kit (Agilent Technologies). Final Gene Expression and Cell Surface Protein libraries were amplified 14 and 10 cycles respectively and visualized on an Agilent TapeStation 4200 using High Sensitivity D1000 ScreenTape (Agilent Technologies). Individual libraries were diluted to 2nM and pooled for sequencing. Pools were sequenced with an S2 100 cycle run kit (28bp Read1, 8bp Index1 and 91bp Read2) on the NovaSeq 6000 Sequencing System (Illumina).