Illumina HiSeq 4000 sequencing; scRNA-seq of lung CD140a+ mesenchymal cells from AT3-gcsf tumor-bearing mice
scRNA-seq of lung CD140a+ mesenchymal cells from AT3-gcsf tumor-bearing mice
The 10-week old female heterozygous mice (B6.129S4-Pdgfratm11(EGFP)Sor/J) used in this study carried one pdgfrα-GFP allele (which does not encode for active PDGFR-α) and one functional pdgfrα allele. AT3-gcsf tumor-bearing CD140a-EGFP mice were euthanized, and lung was harvested and digested. Lung single cell suspension was prepared and stained with primary-fluorophore conjugated antibodies. Lung CD140a+ mesenchymal cells (CD45-CD31-CD326-GFP+) were isolated by fluorescence-activated cell sorting. Cells capture, lysis and RNA extraction were performed using the 10x V3 Chromium technology. Around 12,000 cells were loaded in each lane of the 10x Chromium chip. Barcoding and library preparation were performed with the 10x Chromium system using version 3 chemistry, following the manufacturer's protocol. cDNA was synthesized and amplified as per manufacturer's protocol and amplified to construct an Illumina sequencing library. cDNA and libraries were checked for quality on an Agilent 4200 TapeStation, quantified by KAPA qPCR.