title: GSM6670674: replicate 2 fixed pupal hemocytes, 3 chips; Drosophila melanogaster; RNA-Seq
library_construction_protocol: 50-60 2-10h APF pupae were collected and washed in 1 x PBS. The pupae were then transferred to 1 x Schneider´s Drosophila medium (Gibco) supplemented with 10% FBS, 50 units/mL penicillin, and 50 µg/mL streptomycin and opened using forks to rinse out the hemolymph. Cells were filtered through a 50-µm cell strainer and collected in a total volume of 500 µL. Subsequently, centrifugation at 500 x g and 4°C for 20 minutes was performed. The supernatant was discarded, and the pellet was resuspended with 500 µL PBS + 0.01 % BSA for single-cell sequencing of live cells. For fixed samples, the previous pellet was resuspended with 100 µL 1 x PBS and subsequently fixed by adding 4 volumes of ice-cold 100% methanol (final concentration of 80% methanol in PBS) and thoroughly mixed with a pipette. Cells were stored at - 20°C until use (1-3 days). For single-cell sequencing, cells were moved to 4°C and kept on ice throughout the procedure. Fixed cells were pelleted at 500 x g for 15 minutes and rehydrated in 500 µL PBS + 0.01 % BSA. For datasets 1 and 2 we used fixed samples, while we used live cells for dataset 3. 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 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).