title: NextSeq 500 paired end sequencing; Single-cell RNA-seq of Drosophila embryos to investigate gene expression during dorsal-ventral patterning
design: Single-cell RNA-seq of Drosophila embryos to investigate gene expression during dorsal-ventral patterning
library_construction_protocol: For single-cell RNA-seq, we adapted the collection and methanol fixation procedures described in (Alles et al., 2017; Karaiskos et al., 2017). Following a pre-collection period of at least one hour, fly embryos were collected on yeasted apple juice plates at 25C. After 1 hour of collection, the embryos on the plate were incubated at 25C for 2.25 hours. Embryos were dechorionated for 2 min in 2.6% sodium hypochlorite, rinsed with water, and suspended in PBS, 0.5% Triton X-100. Embryos were rinsed with cell culture grade DPBS without Ca2+ and Mg2+ to remove residual detergent, and placed on ice at precisely 3.5 hours after the start of collection. Embryos were resuspended in 500 ul ice cold dissociation buffer (cell culture grade DPBS without Ca2+ and Mg2+, 0.04% BSA) and dissociated with a clean metal pestle. Cells and tissue fragments were pelleted at 500 x g for 5 min at 4C, then gently resuspended in 100 ul Trypsin-EDTA 0.25% and incubated for 3 minutes. After 3 minutes Trypsin was quenched by adding 1 mL cell culture grade DPBS without Ca2+ and Mg2+, 10% FCS. Cells were pelleted at 1000 x g for 5 min at 4C, then resuspended in 500 ul dissociation buffer, pelleted again and resuspended in 100 ul dissociation buffer. A 10 ul aliquot of cells was kept and counted using an improved Neubauer chamber or a Luna2 cell counter. To fix cells, 4 volumes of 100% methanol, pre-chilled at -20C, were slowly added to the cells. Fixed cells were stored at -80C and used within 3 days. Methanol-fixed cells were spun at 3000 x g at 4C for 5 min and resuspended in 500 ul DPBS + 0.04 % BSA to rehydrate. Rehydrated cells were counted using a Luna2 cell counter, and the volume used for library preparation was chosen for a targeted recovery of 5000 cells. We performed scRNA-seq using the 10X Genomics Chromium Single Cell 3' Reagents v3 (10X Genomics PN-1000075), according to the manufacturer's instructions (Rev B). We performed scRNA-seq library preparation using the 10X Genomics Chromium Single Cell 3' Reagents v3 (10X Genomics PN-1000075), according to the manufacturer's instructions (Rev B).