GSM8702997: APOE4, Female, 3M, #2; Mus musculus; RNA-Seq
The mice were perfused with phosphate-buffered saline (PBS, pH 7.4) after anesthetization with isoflurane, followed by a quick dissection of cortex from left hemisphere to the HBSS buffer (Gibco, 14025076). The cortices were then dissociated with papain-based the Neural Tissue Dissociation kit (Miltenyi Biotec, 130–092-628) using gentleMACS™ Dissociator (Miltenyi Biotec, 130-093-235) followed by debris removal through filtering with the 70-µm cell strainer. Cell suspensions were then subject to myelin removal (Cat# 130-096-433, Miltenyi Biotec), red cell lysis (Cat# 130-094-183; Miltenyi Biotec) and dead cell removal (Cat# 130-090-101; Miltenyi Biotec) according to the manufacturer's instructions. Finally, cell pellets were resuspended in Ca/Mg-free PBS supplemented with 0.5% FBS. Cell numbers and viability were determined through trypan blue examination under microscope. More than 90% of the cells were identified viable for all the 24 samples. The isolated cells were subject to single cell RNA sequencing (scRNAseq) through the chromium platform (10x Genomics) with the 39 gene expression V3 kit, using a targeted input of ∼6,000–9,000 cells per sample. In brief, gel-beads in emulsions (GEMs) were generated on the sample chip in the Chromium controller. Barcoded cDNA was extracted from the GEMs using Post-GEM RT-cleanup and amplified for 12 cycles. Amplified cDNA was fragmented and subjected to end-repair, poly-A-tailing, adaptor ligation, and 10x-specific sample indexing following the manufacturer's protocol. Libraries were quantified using the TapeStation (Agilent) analysis and then sequenced on a HiSeq 4000 instrument (Illumina) targeting a depth of 25,000–50,000 reads per cell.