Apc pdgfrβ

Immediately after dissociation, cells were resuspended in FACS buffer (5% FBS in PBS) and Fc-blocked for 10 min. Benign-enriched tissue from ICC1, ICC3, and ICC6 and tumor-enriched tissue from ICC3 had cell yields below the threshold for sorting on multiple gates, thus only viable cells from these samples were sorted with BD FACSAria III (flow rate 1.0, efficiency >90%) after staining dead cells with DAPI (Invitrogen, #D3571, dilution 1:100) in the dark for 15 minutes at room temperature. For other patient samples, cells were stained in the dark for 30 minutes at room temperature with APC-PDGFRβ (BioLegend; #323608; clone 18A2; dilution 1:80), BV711-Ep-CAM (BioLegend; #324239; clone 9C4; dilution 1:2,500), PE/Cy7-CD45 (BioLegend; #304015; clone HI30; dilution 1:5,000) and washed 3 × 5 minutes with FACS buffer. DAPI (Invitrogen; #D3571; dilution 1:100) was then added to stain dead cells, and viable cells were sorted into EpCAM⁺ (epithelial), CD45⁺ (immune), and others (TME) populations (Supplementary Fig. 10a). BD FACS Diva 8.0.1 software was used to analyze FACS data.

We performed flow cytometry to confirm typical markers expressed by pericytes. Single cell suspensions of pericytes were stained for 30 min at room temperature (RT) using fluorescence-labelled antibodies for pericyte surface markers (488-NG2 from Invitrogen, APC PDGFR-β and APC CD105 from Biolegend) and DAPI for cell viability. Labelled cells were then washed in PBS + 1% BSA and were analyzed on a FACSCanto II flow cytometer (Becton Dickinson). Data analysis was performed using FlowJo software (Tree Star, v10).

E11 embryos were obtained from Runx1-GFP/+ mice mating and AGM cells stained with NG2 Cy3 (1:100, Millipore, ab5320c3), PDGFRβ APC (1:100, Biolegend, 136008), CD31 PECy7 (1:4000, eBioscience, 25-0311-82), CD45 PerCypCy5.5 (1:400, BD Pharmingen, 550994) and ckit BV421 (1:500, BD Horizon, 562609) for 30 min at 4 °C, washed, centrifuged, then resuspended in PBS/FCS/PS for analysis and cell sorting. Cells were purified using BD FACS ARIA Fusion cell sorter (BD Bioscience) and Sytox AAD was used to select viable cells. Cells were sorted and collected directly into 10–20 µl of lysis buffer containing Nuclease-free water (Ambion AM9930), 0.2% Triton and 1/20 RNAse inhibitor (Thermoscientific 10777019). Full-length cDNA was generated from 2.3 µl of this cell lysate using the Smarter2 procedure as described^{75 (link)}. Sequencing libraries were generated from 500 pg of cDNA with Illumina’s Nextera XT sample prep kit (Illumina Inc., U.S.A) and according to the Illumina TruSeq Rapid v2 protocol on the HiSeq2500 with a single read 51 bp and dual 9 + 9 bp index (Illumina Inc., U.S.A). Reads were aligned against the GRCm38 reference using HiSat2 (version 2.0.4). We called gene expression values using GENCODE M19 gene annotation file and the union mode in the Bioconductor Genome Alignments Package (v1.8.1).