Deltagene assays

The Fluidigm BioMark 96.96 Dynamic Array (28 (link)) was used to measure the gene expression in small cell populations. Ten cells per well were sorted by FACS in quadruplicate into 96-well plates containing a reaction mix for reverse transcription (CellsDirect One-Step qRT-PCR kit; Invitrogen) and pre-amplification with 96 selected gene primer pairs (DELTAgene assays, Fluidigm). After sorting, samples were reverse-transcribed and pre-amplified for 18 cycles. Primers and dNTPs were removed by incubation with ExonucleaseI (NE Biolabs), and samples were diluted (5×) with TE buffer and stored at −20°C. Samples and assays (primer pairs) were prepared for loading onto 96.96 Fluidigm Dynamic arrays according to the manufacturer’s recommendations. The 96.96 Fluidigm Dynamic Arrays (Fluidigm Corp.) were primed and loaded on an IFC Controller HX (Fluidigm Corp.) and real-time PCR was run on a BiomarkHD (Fluidigm Corp.). Data were collected and analyzed using Fluidigm Real-Time PCR Analysis software (v4.1.2).

Single embryonic lung epithelial cells were captured on a medium-sized (10-17 µm cell diameter) microfluidic RNA-seq or STA chip (Fluidigm) using the Fluidigm C1 system. To ensure unbiased and comprehensive profiling all distal lung epithelial cells, an initial experiment was performed using a microfluidic chip with a 17-25µm capture range; however no cells with diameter greater than ~15 µm were captured indicating that no major cell populations were missed by using the smaller capture range (Extended Data Figure 2b). Cells were loaded onto the chip at a concentration of 300-500 cells/µl, stained for viability (LIVE/DEAD cell viability assay, Molecular Probes, Life Technologies) and imaged by phase-contrast and fluorescence microscopy to assess number and viability of cells per capture site. Only single, live cells were included in the analysis. For RNAseq experiments, cDNAs were prepared on chip using the SMARTer Ultra Low RNA kit for Illumina (Clontech). ERCC (External RNA Controls Consortium) RNA spike-in Mix (Ambion, Life Technologies) was added to the lysis reaction and processed in parallel to cellular mRNA. For qPCR experiments, amplicons were prepared using pooled DELTAgene assays (Fluidigm) and Ambion (Life Technologies) Cells to CT lysis and pre-amplification kit using the protocol provided by Fluidigm.