GSM8194569: single-embryo Library 2; Drosophila melanogaster; OTHER
Frozen embryos or unfertilized eggs were homogenized in 1 mL 80% methanol (v/v) (Optima™ LC/MS Grade, Fisher Chemical™) by bead-beating at 6 m/s for 30 seconds at 4°C using a Bead Ruptor Elite homogenizer coupled with Bead Ruptor Cryo Cooling Unit (OMNI international, USA). After metabolite extraction, the pellet, ceramic beads, and remaining metabolite soluble fraction (~200 µL) of each sample was dried (~1 h) in a Genevac EZ-2 series evaporator using low-BP lamp-off settings. RNA was then isolated following a miniaturized version of the TRIzol protocol. Briefly, 500 µL TRIzol™ (Invitrogen, USA) and 50 µL Gibco™ phosphate buffered saline pH 7.4 (Thermo Fisher Scientific, USA) were added to each sample mixing thoroughly. After 5 min incubation at room temperature, 100 µL chloroform (Sigma-Aldrich, USA) were added, samples mixed by vortex, incubated 2 min at room temperature, centrifuged for 15 minutes at 12,000 × g at 4 °C, and RNA-containing aqueous phase pipetted into a new 1.5 mL tube. RNA was precipitated by adding 250 µL ice-cold isopropanol (Sigma-Aldrich, USA) and 2 µL GlycoBlue™ (Thermo Fisher, USA), samples mixed by hand, incubated for 10 min at RT, and centrifuged for 10 minutes at 12,000 × g at 4°C. After discarding the supernatant, RNA pellets were washed with 1 ml 75% (v/v) ethanol (Pharmco, USA), air-dried, and stored at -80 °C until library preparation for RNA-seq. RNA-seq was carried out following a miniaturized version of the sensitive highly-multiplexed single-cell RNA-seq (CEL-Seq2) protocol (Hashimshony et al. 2016) using the I.DOT liquid handler (Dispendix). Dried RNA was resuspended in 8 µL nuclease-free water (Invitrogen, USA) and 120 nL dispensed into a 384-well plate holding 240 nL of primer-mix including 192 different cell barcodes with unique molecular identifiers (UMI). Subsequent steps and reagents are detailed in Pérez-Mojica et al., 2023. DOI:10.1016/j.xpro.2023.102535, except that libraries were diluted 1:10 (cDNA:H2O) before an 11-cycle amplification. Each library includes 96 single-embryos. Modified CelSeq2 protocol