Nucblue cell stain reagent

Samples were prepared, processed and analyzed for single cell transcriptomics at NIG, Institute of Human Genetics, University Medical Center Göttingen. Briefly, cells were distributed on 5,184 nanowell chips ICELL8 250v Chip (ICELL8 System, Takara Bio). Single alive cells were identified using Hoechst 33342 and propidium iodide staining (NucBlue Cell Stain Reagent, Thermo Fisher Scientific) and the CellSelect Software (Takara Bio). Complementary DNA synthesis was performed by oligo-dT priming in a one-step RT-PCR reaction. P5 indexing primers, Terra Polymerase and Reaction Buffer were added for library preparation. Transposase enzyme and reaction buffer (Tn5 mixture) were added to each well. P7indexing primers were dispensed to wells. Final libraries were amplified and pooled as they are extracted from the chip. Pooled libraries were purified and size selected with Agencourt AMPureXP magnetic beads (Beckman Coulter) to obtain an average library size of 500 bp. Libraries were sequenced with a HiSeq4000 (Illumina) to obtain on average ~3·10⁵ reads per cell (single-end, 50 bp).

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).

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.