GSM9896010: 16PlexMs_FLEX_Cells_GEX; Mus musculus; RNA-Seq
Mice were deeply anesthetized and perfused intracardially with ice cold PBS, brains were removed, and hippocampi were quickly dissected on ice. We collected hippocampi from 2 brains/sample. The cell suspensions were prepared using the Adult Brain Dissociation Kit (Miltenyi Biotec 130-107-677) according to the manufacturer's instructions with the following modifications that increased cell yield. The tissues were chopped into small pieces on ice prior to adding to the Enzyme 1 mix, after enzymatic and mechanical dissociation, the mixtures were triturated with P1000 5-10 times to dissociate undigested pieces of tissue, red blood cell removal step was omitted since brains were thoroughly perfused with PBS. Cell counts and viability were measured with AO/PI staining in an automated cell count system (Cellometer Cell Viability Counter, Nexcelom). Cells were immediately fixed using 10X FLEX reagents and 10x Genomics protocol, counted post-fixation and stored at -80 C according to 10X Genomics protocol. 4 biological replicates per genotype (M:F ratio was 40:60), each replicate containing hippocampi from two brains, were submitted to the Single Cell Transcriptomics Core at the University of Pittsburgh. Each sample contained ∼300,000 cells and the viability was >80%. Libraries were prepared using the Chromium Fixed RNA Profiling Reagents Kit for Multiplexed Samples (10x Genomics) according to the manufacturer's protocol. Libraries sequencing performed by the Health Sciences Sequencing Core at the University of Pittsburgh generated 1.8 billion reads which covers more than 10,000 reads per cell.