Embryonic cortices at E14 and E18 were transferred to dissociation media: HBSS (Life Technologies), 10 mM Hepes (Sigma), 172 mg/L kynurenic acid (Sigma), 0.86 g/L MgCl2·6H2O (Sigma), and 6.3 g/L D-glucose (Sigma). This solution was saturated with 95% oxygen and 5% CO2 and was pH-adjusted to 7.35 before use. Papain (20 U/mL; Worthington), Pronase from Streptomyces griseus (1 mg/mL; Sigma), proteinase XXIII from Aspergillus melleus (3 mg/mL; Sigma), and DNase (2 mg/mL; Worthington) were added to the dissociation media. Dissociation was carried out at 37 °C for 1 h. The samples were then triturated, filtered, and spun at 300 × g for 5 min. The pellet was resuspended in trypsin inhibitor solution (dissociation media with 1% BSA and 1% ovomucoid). Following gradient centrifugation (70 × g for 10 min), the cells were washed in dissociation media containing 0.04% BSA and resuspended in dissociation media containing 0.04% BSA and 15% OptiPrep (Sigma). Individual cells were captured and barcoded using the inDrops platform as previously described.30 Briefly, single-cell suspensions were fed into a microfluidic device that packaged the cells with barcoded hydrogel microspheres and reverse transcriptase/lysis reagents. After cell encapsulation, primers were photo-released by UV exposure. Indexed libraries were pooled and sequenced on a NextSeq 500 (Illumina). Individual cells were captured and barcoded using the inDrops platform as previously described. Briefly, single-cell suspensions were fed into a microfluidic device that packaged the cells with barcoded hydrogel microspheres and reverse transcriptase/lysis reagents. After cell encapsulation, primers were photo-released by UV exposure. Two libraries of approximately 5000 cells each were collected for each sample. Indexed libraries were pooled and sequenced on a Nextseq 500 (Illumina).
GEO Accession
GSM4456522