GSM6307659: PBS_lin-neg_Mouse_1; Mus musculus; RNA-Seq
GSM6307659
Cells were counted and stained with anti-mouse Epcam CD326-AlexaFluor647 antibody (1:100, Biolegend, 118212), CD31-APC (1:250, Invitrogen, 17-0311-82), CD45-APC (1:250, Biolegend, 103112), Lyve1-AF647 (Invitrogen, 50-0443-82), Ter119-APC (1:100, Biolegend, 116218), and Zombie Violet for viability (BioLegend, 423113). All stainings were performed per 10 MIo cells in 1ml. Cells were stained with Zombie violet according to manufacturer's instructions for 15 min at RT, washed once with PBS and  blocked with anti-mouse FC block for 10min on ice. Staining occurred for 20min at 4°C in the dark in the FACS buffer (PBS + 0.1% BSA), and cells were washed once and filtered with a cell strainer (70µM)  before subjecting them to FACS sorting. Mesenchymal cells were sorted as ZombieViolet-/APC- and TCF21 lineage was distinguished by the dt-Tomato signal in the PE channel. Data acquisition was performed in a BD Fortessa flow cytometer (Becton Dickinson, Heidelberg, Germany). 30.000 cells per lineage and mouse were sorted into cooled RPMI media. The sorted cells were were diluted to 1000 cells/µl, and loaded on the 10x Chromium Next GEM Chip G with a targeted cell recovery of 10,000. The following steps were completed according to the manufacturer's protocol (Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1). Libraries have been pooled according to their minimal required read counts (35,000 or 50,000 reads/cell for 3′ gene expression libraries, 20,000 reads/cell for 5′ gene expression libraries, and 5000 reads/cell for TCR libraries). Illumina paired-end sequencing was performed with 150 or 200 (3′ gene expression) on a NovaSeq 6000.