Cellselect software

The Takara ICELL8 5,184 nano-well chip was used with the full-length SMART-Seq ICELL8 Reagent Kit. Cell suspensions were fluorescent-labelled with live/dead stain, Hoechst 33,342 and propidium iodide (NucBlue Cell Stain Reagent, Thermo Fisher Scientific) for 15 min prior to their dispensing into the Takara ICELL8 5,184 nano-well chip. CellSelect Software (Takara Bio) was used to visualize and select wells containing single and live cells. Next, cDNA was synthesized via oligo-dT priming in a one-step RT-PCR reaction. P5 indexing primers for subsequent library preparation were dispensed into all wells receiving a different index, in addition to Terra polymerase and reaction buffer. Transposase enzyme and reaction buffer (Tn5 mixture) were dispensed to selected wells. P7 indexing primers were dispensed to wells. Final Illumina libraries were amplified and pooled as they were extracted from the chip. Pooled libraries were purified and size selected using Agencourt AMPure XP magnetic beads (Beckman Coulter) to obtain an average library size of 500 bp. A typical yield for a library comprised of ~ 1,300 cells was ~ 15 nM. Libraries were sequenced on the HiSeq 4000 (Illumina) to obtain on average ~ 0.3 Mio reads per cell (SE; 50 bp).

Single-cell RNA-seq of tdTomato+ (hereafter referred to as tdTom+) cells isolated from Acta2-Cre-ERT2; tdTomato^flox lungs was performed using the ICELL8 platform (Takara Bio) using the V2 protocol with in-chip preamplification. Samples were presorted into HBSS (Gibco) at a concentration of 20 cells/µL and stained with Hoechst dye (Invitrogen) for 5 min at RT prior to loading onto the MSND dispenser. For each sample, 3 dispenses were made on one 5,184-well chip resulting in 348 and 459 wells containing single cells after selection in CellSelect software (Takara Bio). Selected wells were processed for RT and in-chip preamplification, and cDNA was pooled for final library preparation using standard Nextera protocol (Illumina). The library was checked in Labchip Gx touch 24 (Perkin Elmer) and sequenced on Nextseq500 (Illumina) using V2 chemistry and paired-end setup (11 bp Read1 for cell barcode and 80 bp Read2 for cDNA template sequencing). A total of 537 M reads were obtained.
Single-cell RNA-seq of tdTom+ cells isolated from Gli1^Cre−ERT2; tdTomato^flox lungs was carried out using the 10x Genomics Single-Cell 3′ v3.1 RNA-seq kit. Gene expression libraries were prepared according to the manufacturer’s protocol. MULTI-seq barcode libraries were retrieved from the samples, and libraries were prepared independently.

The Takara ICELL8 5184 nano-well chip was used with the full-length SMART-Seq ICELL8 Reagent Kit. Cell suspensions were fluorescently labeled with live/dead stain, Hoechst 34580 (Thermo Fisher Scientific) and propidium iodide (NucBlue Cell Stain Reagent, Thermo Fisher Scientific) for 15 min prior to being dispensed into the Takara ICELL8 5184 nano-well chip. CellSelect Software (Takara Bio) was used to visualize and select wells containing single and live cells. Next, cDNA was synthesized via oligo-dT priming in a one-step RT-PCR reaction. P5 indexing primers for subsequent library preparation were dispensed into all wells receiving a different index, in addition to Terra polymerase and reaction buffer. Transposase enzyme and reaction buffer (Tn5 mixture) were dispensed to selected wells. P7 indexing primers were dispensed to wells. Final Illumina libraries were amplified and pooled as they were extracted from the chip. Pooled libraries were purified and size selected using Agencourt AMPure XP magnetic beads (Beckman Coulter) to obtain an average library size of 500 bp. A typical yield for a library comprising ∼1300 cells was ∼15 nM. Libraries were sequenced on the HiSeq 4000 (Illumina) to obtain on average ∼0.3 Mio reads per cell (SE; 50 bp). A list of Gal4-enhancer trap and GFP-exon trap fly lines used for ex vivo validation can be found in Table S19.