Dnase

Isolation of SFs was performed from both hind paws. The ankle joints were dissected, and the tissues were disaggregated by incubation for 30 min at 37 °C in an enzymatic digestion medium consisting of DMEM, 10%heat-inactivated FBS, collagenase (0.5 mg ml⁻¹) from Clostridium histolyticum (Sigma, C5138) and 0.03 mg ml⁻¹ DNase (Sigma, 9003-98-9). Upon washing the cells with PBS containing DNase, they were blocked in 1% BSA in PBS and Fc blocker (unlabelled anti-CD16/32, Biolegend 101302) for 10 min at 4 °C and stained with fluorophore-conjugated antibodies for 20 min at 4 °C (anti-Pdpn PE-Cy7, Biolegend 127411; anti-Thy1 A647, Biolegend 105318; anti-CD31 APC/Fire 750, Biolegend 102433; anti-CD45 APC-Cy7, Biolegend 103116; anti-Ter119 APC-A780, eBioscience 47-5921-80). After washing with PBS, cells were resuspended in FACS buffer (PBS, 1%BSA). Sorting of cells was performed with BD FACSAria III and the BD FACSDiva software, and dead cells were excluded by DAPI staining. Sorting gaiting for single-cell RNA-seq/ATAC-seq and bulk RNA-seq was different (Additional file 1: Fig. S1B). For sorted populations, purity and viability were determined by reanalysis for the target population based on cell surface markers immediately post-sorting. Purity was > 99% for each target population.

Whole-lung single-cell suspensions were prepared as above and then blocked in SB containing 1:50 TruStain FcX (anti-mouse CD16/32) Antibody (BioLegend, #101319) for 10 min at 37°C. The cell suspension was stained using allophycocyanin/Cy7-conjugated rat anti-mouse CD45 antibody (1:200, BioLegend, #101319), PE-conjugated rat anti-mouse EpCam antibody (1:500, BioLegend, G8.8, #118206) for 45 min at 4°C. Stained cells and ‘fluorescence minus one’ controls were then resuspended in SB + 1:1000 Dnase + 1:1000 Draq7 (BioLegend, #424001) as a live/dead stain. All FACS sorting was done on a BD FACSAria Fusion Sorter (BD Biosciences).

Whole lung single-cell suspensions were prepared as above and then blocked in SB containing 1:50 TruStain FcX (anti-mouse CD16/32) antibody (BioLegend, #101319) for 10 min at 37°C. The cell suspension was stained using allophycocyanin (APC)/Cy7–conjugated rat anti-mouse CD45 antibody (1:200; BioLegend, #101319), Alexa Fluor 488–conjugated rat anti-mouse CD31 [platelet endothelial cell adhesion molecule 1 (PECAM1)] antibody (1:200; BioLegend, MEC13.3, #102513) for 45 min at 4°C. Stained cells and “fluorescence minus one” controls were then resuspended in SB + 1:1000 DNase + 1:1000 Draq7 (BioLegend, #424001) as a live/dead stain. All FACS sorting was performed on a BD FACSAria Fusion Sorter (BD Biosciences).