GSM8005188: Sample 11 cortex, Emx1CreERT2;Ash1lfl/fl, scRNA-seq; Mus musculus; RNA-Seq
Embryonic cortices were dissected in Earle's balanced salt solution (EBSS) and incubated with papain, 5.5 mM L-cysteine-HCL, 1.1 mM EDTA, and 100 mg/ml DNase I (all part of Papain Dissociation System. Worthington Biochemical Corporation, NJ, USA; cat: LK003153) in O2:CO2 equilibrated EBSS for 8 min at 37°C. Samples were titrated and centrifuged. Cells were resuspended with ovomucoid protease inhibitor and 50 mg/ml DNase I in O2:CO2 equilibrated EBSS and passed through a 70μm cell strainer (Becton Dickson, NJ, USA; cat: 08-771-2). Cells underwent centrifugation with an ovomucoid protease inhibitor gradient and then resuspended in N2/B27 media (all from Invitrogen, MA, USA. Neurobasal media cat: 21103-049; B27 supplement cat: 17504044; L-glutamine cat: 25030-081). Cells were counted before subsequent scRNA sequencing steps. Single cell RNA sequencing was carried out using the Seq-Well platform (Gierahn et al, 2017). Briefly, functionalized Seq-Well arrays were loaded with mRNA Capture Beads (ChemeGenes, Wilmington, MA; cat: MACOSKO-2011-10). Cells were loaded on the arrays and incubated for 15 min. Functionalized membranes (custom ordered from Sterlitech, WA, USA) were sealed onto the arrays and incubated at 37°C for 45 min. Sealed arrays underwent incubations in lysis and hybridization buffers. Beads were removed from the arrays via centrifugation. Beads were incubated with Maxima Reverse Transcriptase (Thermo Fisher Scientific, MA, USA; cat: EPO0753) overnight, then treated with Exonuclease 1 (New England Biolabs, MA, USA; cat: M0293S). Transcriptome amplification was performed using the 2X KAPA Hifi Hotstart Readymix (Roche, Basel, Switzerland; cat: KK-2602). Products were purified with AMPure SPRI beads (Beckman Coulter, CA, USA; cat: A63881). Libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina, CA, USA; cat: FC-131-1096) and libraries were sequenced on an Illumina NextSeq 2000 instrument. Digital gene expression matrices were generated from sequencing data using the Drop-seq Alignment protocol (Macosko et al, 2015). Reads were aligned to mm10 with STAR.