概要: Our work illustrates how high-resolution molecular and spatial profiling of COVID-19 patient tissues collected during rapid autopsies can serve as a hypothesisgenerating tool to identify key mediators driving the pathophysiology of COVID-19 for diagnostic and therapeutic target testing. Here we employ bulk RNA sequencing to identify key regulators of COVID-19 and list specific mediators for further study as potential diagnostic and therapeutic targets. We use single-nuclei RNA sequencing to highlight the diversity and heterogeneity of coronavirus receptors within the brain, suggesting that it will be critical to expand the focus from ACE2 to include other receptors, such as BSG and ANPEP, and we perform digital spatial profiling of lung and lymph node tissue to compare two patients with different clinical courses and symptomatology.
项目整体设计: Two technical replicates across ten tissues (Patient 1) with two technical replicates of a pool of brain RNA from multiple donors as control (no SARS-CoV-2 infection).
Total RNA was extracted from the autopsy tissues by first flash freezing each tissue in liquid nitrogen and then using the CryoPREP Tissue Disruption System (Covaris) to pulverize the tissue into a fine powder. The powder was then immediately resuspended in cell lysis buffer and RNA was extracted using the ReliaPrep RNA Tissue Miniprep System (Promega, #Z6112) by following the manufacturer’s instructions. A DNase digestion was also performed during the RNA extraction workflow to remove any potential contamination from carryover genomic DNA (reagents included in the kit). The extracted RNA was then purified using 1.8X RNAClean XP beads (Beckman Coulter) to remove any molecular impurities.
建库方案:
Stranded total RNA libraries were generated according to the manufacturer's instructions, using the TruSeq Stranded Total RNA Gold kit for Illumina sequencing
