title: GSM4848459: Control Sample 8; Homo sapiens; RNA-Seq
library_construction_protocol: Frozen medial frontal cortex tissue from post-mortem control and COVID-19 patients were obtained from the Stanford/ VA/ NIA Aging Clinical Research Center (ACRC) and the Saarland University Hospital Institute for Neuropathology, with approval from local ethics committees. The protocol for the isolation of nuclei was adapted from previous studies, and performed in a BSL2+ Biosafety Cabinet wearing PPE (Mathys et al., Nature 2019). All procedures were carried out on ice or at 4°C. Briefly, 50 mg of post-mortem brain tissue was dounce homogenized in 2 ml of Nuclei EZ Prep Lysis Buffer (Sigma, NUC101) spiked with 0.2 U μl−1 RNase inhibitor (Takara, 2313A) and EDTA-free Protease Inhibitor Cocktail (Roche, 11873580001) before incubating on ice for 5 min in a final volume of 5 ml. Homogenized tissue was filtered through a 100-μm cell strainer (Falcon, 352360), mixed with an equal volume of 50% iodixanol density gradient medium in PBS (OptiPrep, Sigma-Aldrich, D1556) to make a final concentration of 25% iodixanol. 30% iodixanol was layered underneath the 25% mixture. Similarly, 40% iodixanol was layered underneath the 30% iodixanol. In a swinging-bucket centrifuge, nuclei were centrifuged for 20 min at 3,000 r.c.f. After centrifugation, the nuclei were present at the interface of the 30% and 40% iodixanol solutions. Isolated nuclei were resuspended in 1% BSA with 0.2 U μl−1 RNase inhibitor, filtered twice through a 40 μm strainer (Flowmi), and counted on an TC20 automated cell counter (Bio-Rad) after the addition of Trypan Blue.    Frozen choroid plexus tissue was extracted from the lateral ventricles of post-mortem tissue obtained from the Stanford University Pathology department and the Saarland University Hospital Institute for Neuropathology, with approval from local ethics committees. All procedures were carried out on ice or at 4°C, and in a BSL2+ Biosafety Cabinet wearing PPE. Dounce homogenization or enzymatic dissociation successfully used for mice choroid plexi (Dani et al., bioRxiv 2019) resulted in loss of nuclei integrity and low nuclei complexity (<50 median genes/ nuclei). We hypothesized that like shaking an apple tree, gentle pipetting of choroid plexi tissue in lysis buffer could liberate nuclei without needing to physically disintegrate the fibrous choroid matrix—and thus avoid collateral physical damage to nuclei. Specifically, 40 mg sof choroid plexus tissue was thawed in 250 μl of 1% BSA with 0.2 U μl−1 RNase inhibitor until the tissue settled. 5 ml of lysis buffer (10 mM Tris, 10 mM NaCl, 3 mM MgCl2, 0.1% Nonidet P40 substitute (Roche/ Sigma, 11754599001), 0.2 U μl−1 RNase inhibitor, and protease inhibitor) was added and tissue incubated on ice for 10 minutes with gentle swirling every 2 minutes. 5 ml of 1% BSA was added and the tissue triturated 10 times with a 5 ml serological pipette. After centrifugation (500g, 5 min), pelleted nuclei were resuspended in 1% BSA with 0.2 U μl−1 RNase inhibitor; gently triturated 10 times with a 1 ml regular-bore pipette tip; and filtered twice through a 70 μm and then a 40 μm strainer (Flowmi). Debris was inspected on a brightfield microscope and nuclei were counted on an TC20 automated cell counter (Bio-Rad) after the addition of Trypan Blue. For droplet-based snRNA-seq, libraries were prepared using the Chromium Next GEM Single Cell 3ʹ v3.1 according to the manufacturer's protocol (10x Genomics), targeting 10,000 nuclei per sample after counting with a TC20 Automated Cell Counter (Bio-Rad). 13 cycles were applied to brain parenchyma samples to generate cDNA, and 15 for choroid plexus samples. All samples underwent 15-16 cycles for final library generation. Generated snRNA-seq libraries were sequenced across two S4 lanes on a NovaSeq 6000 (150 cycles, Novogene).
attributes.tag: GEO Accession
attributes.value: GSM4848459