Influx machine

Human PDGFRα + cMSCs were isolated by explant culture and purified by fluorescence-activated cell sorting as previously described^{17 (link)}. Briefly, frozen left ventricular samples (~100–200 mg) were removed from liquid nitrogen and quickly thawed by washing twice with ice cold DPBS solution. The tissue was minced into small segments and then placed in 6-well plates coated with 0.1% (wt/vol) gelatin. All explants were cultured in MEMα medium (Sigma-Aldrich) supplemented with 20% FBS (Sigma-Aldrich), 2 mM L-glutamine and 1x penicillin-streptomycin (Sigma-Aldrich), in a 5% CO₂ humidified incubator at 37 °C. The culture media was replaced every 2–3 days. After 2–3 weeks, the outgrowth of cells was dissociated with trypsin-EDTA (Sigma-Aldrich) and stained with anti-PDGFRα antibody-APC (R&D; 1:10), anti-CD31-PE (R&D; 1:20) and anti-CD90-FITC (BioLegend; 1:20). The cells were then sorted for PDGFRα+/CD90+/CD31⁻ fraction using Influx machine (BD Biosciences), as described previously^{17 (link)}.

Ten to twelve E11.5 cortices, spinal cords, stomachs and lungs/esophaguses were dissociated using a Miltenyi Biotec Neural Dissociation Kit (P; #130-092-628). 63892 cortex, 59193 spinal cord, 72151 stomach and 40074 lung/esophagus cells were then FACS sorted into triplicates on a BD Influx machine, with plots shown in S1 Fig. RNA was extracted using a Qiagen RNeasy mini kit, cDNA was made using the Smartseq2 protocol [43 (link)], and libraries were produced by following the Nextera XT manufacturer’s instructions. Sequencing of 50bp single end reads was done using an Illumina Genome Analyzer IIx. Star v2.5 [44 (link)] was used to align reads to mm9, while gene expression levels were calculated using rpkmforgenes.py [45 (link)]. Differential gene expression was assessed using Deseq2 [46 (link)], with organ specific genes showing differential expression against all other triplicate samples padj < 0.01 and fold change > 2. PCA was performed using Rstudio Prcomp.

Cells were collected by trypsinisation and analysed using a FACS Calibur (BD) or an LSR Fortessa (BD). Flow cytometry data was analysed using the FlowJo software package. Cells resuspended in sorting buffer (PBS + 10 mM HEPES +2% FCS) were filtered through a 50 µm filter, and sorted on an Influx machine (BD), or, for the ubiquitome CRISPR/Cas9 screen, on a FACS Melody (BD). Sorted cells were collected in DMEM +50% FCS and subsequently cultured in DMEM +10% FCS+penicillin/streptomycin. For MHC-I flow cytometric analysis, cells resuspended in cold PBS were incubated with W6/32 (20 min, 4°C), washed twice and then incubated with Alexa-647-labelled anti-mouse antibody (15 min, 4°C). Cells were washed twice and resuspended in PBS.