GSM8945747: fAD iNs cultures with WT iAs and WT iMGLs; Homo sapiens; RNA-Seq
Cells were dissociated with a 1:1 mixture of warm trypsin-EDTA:cold StemPro Accutase Cell Dissociation Reagent for 10min at 37°C. Trypsin was quenched with Trypsin Neutralization Solution (ScienCell, Cat# 0113-2) prior to collection of cell suspension. Cell suspensions were centrifuged for 5 min at 300 x g and resuspended in 1 mL of BrainPhys media. The cell pellet was triturated gently with a p1000 tip and passed through a 40µM Flowmi Cell Strainer (Millipore, BAH136800040-50) to remove debris. Cell suspensions were centrifuged at 200 rcf for 5min and resuspended in 0.04% BSA in PBS. Washes were repeated two more times. At the end of the final wash, cell suspension was passed through another 40µM Flowmi Cell Strainer and counted. Each triple-culture was loaded onto an individual 10X Chromium Chip well (~64,000 cells per well) and resulting emulsions were used to generate scRNAseq libraries using the Chromium Next GEM Single Cell 3' v3.1 chemistry. This loading results in a higher percentage of doublets (which are filtered downstream), ultimately yielding ~20,000 recovered singlet cells per 10x well. Libraries were multiplexed and sequenced on a NovaSeq to an average depth of >20K reads per cell based on estimated yields.