GSM8603228: DS18_E18.5; Mus musculus; RNA-Seq
Cortical tissue was dissected from E18.5 mouse embryos in cold Leibovitz's L15 medium supplemented with 7mM HEPES, pooling embryos from the same litter. Tissue was dissociated into single cell suspensions using reagents from the Worthington Tissue Dissociation Kit; tissue was digested in papain/DNAse for 15 min at 37C with carbogen oxygenation, mechanically triturated in EBSS, and resuspended in an optimem-trehalose media (Optimem supplemented with 50mM of trehalose, 10mMglucose, 0.5 mM kynurenic acid, 0.025 mM APV, 4 mM MgCl2, 0.2 mM HEPES, with osmolarity of 290 ± 10 mOsm/kg). Debris was removed with an 5% optiprep gradient spin and the cell pellet was resuspended in 500 uL of optimem-trehalose media. We sorted for live eYFP+ cells using an Astrios EQ sorting flow cytometer with an 100 um nozzle Libraries construction was done with 10x genomics Chromium reagents to load single cells, form GEMs, and perform the RT reaction. Sequencing was done on a Illumina Nextseq200 with a P2 flowcell to a depth of approximately 200 million reads.