Single-Cell Profiling of the Inflammatory Microenvironment in the Aged Rats Hippocampus and the Contributions of Cerebrovascular Endothelial Cells
Single-Cell Profiling of the Inflammatory Microenvironment in the Aged Rats Hippocampus and the Contributions of Cerebrovascular Endothelial Cells
Pair of hippocampi from LPS and NC group rats(n = 3 per group) were harvested on day 3 after LPS injection and pooled by group for scRNA-seq. Briefly, rats were anesthetized and perfused through the heart with ice-cold PBS. The hippocampus was immediately isolated and enzymatically dissociated using the Adult Brain Dissociation Kit according to the manufacturer's instructions (Miltenyi Biotec, 130-107-677). After removal of debris and red blood cells, the suspended single cells were collected in Dulbecco's phosphate buffered saline (DPBS) supplemented with 0.04% bovine serum albumin. Cells were stained with trypan blue and the proportion of viable cells (unstained) was determined using a Luna-FL™ automated cell counter (Logos Biosystems, Anyang, Kyonggi-do, South Korea). Samples with viable cell counts above 85% were loaded onto a 10x Genomics Chromium chip according to the manufacturer's instructions.  Single-cell suspensions were prepared from dissected hippocampal tissues, followed by RNA extraction using the [StepOnePlus Real-Time PCR System] according to the manufacturer's instructions. RNA quality was assessed by Bioanalyzer (Agilent). Single-cell RNA libraries were prepared using the 10x Genomics Chromium Single Cell 3' v3 kit, following the manufacturer's protocol. Briefly, single cells were partitioned into gel beads in emulsion (GEMs), reverse transcription and cDNA amplification were performed, followed by library construction.