GSM5609928: Neural co-culture 2; Homo sapiens; RNA-Seq
For neural co-culture dissociation, cells were dissociated with TrypLE (Gibco) for 5 minutes 37º C. For purified neurons, cells were dissociated with 0.25% Trypsin-EDTA (Gibco) for 5 minutes 37º C. Dissociated cells were washed in PBS+10% FBS and stained with live/dead DAPI (0.5ug/ml) for 10min on ice. Cells were strained with a 40µ filter in flow buffer of PBS+2% FBS and live cells (neural co-culture) and live tdTomato+ cells (purified neurons) were sorted with FACS Aria for library preparation using Single Cell 3' Reagent Kit v3 (10xGenomics, Chromium). Samples for single-cell RNA-seq (scRNA-seq) was examined first for cell viability, density, and prep quality ensuring no clumps, cell debris, etc. Samples passing the QC process were loaded onto 10x Genomics Chromium for partitioning and encapsulation of single cells into nanoliter-sized GEMs (Gel beads-in-EMulsion). Each encapsulated cell was then lysed within its GEM. The total RNA released was reverse transcribed to cDNA with primers attached to the gel bead, each of which carries a unique 10x barcode for downstream cell separation. Subsequently the GEMs were broken, and all uniquely barcoded cDNAs were pooled and then PCR amplified to generate enough material for Illumina sequencing. This sequencing library construction process was carried out using the 10X Genomics Single Cell 3' v3 Protocol. Sequencing of the 10x scRNA-seq libraries was performed on Illumina HiSeq 4000. 10x RNA seq