title: GSM6599296: single-embryo Library 2; Drosophila melanogaster; RNA-Seq
library_construction_protocol: Single embryos were homogenized in 500 µL TRIzol™ (Invitrogen, USA) by bead-beating with 0.2 g lysing matrix D beads (MP Biomedicals, USA) at 6 m/s for 30 seconds using the FastPrep-24™ instrument (MP Biomedicals, USA). RNA was then isolated following a miniaturized version of the manufacturer's instructions. Briefly, 100 µL chloroform (Sigma-Aldrich, USA) was added, samples mixed by vortex, incubated 2 min at room temperature (RT), and centrifuged for 15 minutes at 12,000 × g at 4 °C to recover RNA-containing aqueous phase in a fresh 1.5 ml microtube. 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. RNA pellets were washed with 1 ml 75% (v/v) ethanol (Pharmco, USA), dried and stored at -80 °C until further use. This same protocol was followed to isolate RNA from fixed embryos using 1 ml TRIzol™ and proportional changes in chloroform and isopropanol. 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 (CELLINK). 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 Sagar et al., 2018, except that libraries were diluted 1:10 (cDNA:H2O) before an 11-cycle amplification. Modified CelSeq2 protocol