GSM5068925: covid-19_snRNAseq [LUNG07_IPF]; Homo sapiens; RNA-Seq
snRNA-seq: After collection, freezing of lung autopsy sam-ples was performed as quickly as possible by placing the samples in cryo-tubes, which were sub-sequently snap-frozen with liquid N2 (5 min). Afterwards, the tubes were placed on dry ice and stored at -80°C. For isolation of nuclei, frozen tissue samples were transferred to an Eppendorf tube containing 0.5 mL TST-buffer (comprised of 0.25 mL 2X ST-based buffer (nuclease-free water (VWR #E476), supplemented with 192 mM NaCl (VWR #27810.364), 20 mM Tris-HCl (pH 7.5) (Sigma Aldrich #T2194), 2 mM CaCl2. 42 mM MgCl2 and 0.2 units/ µL RNasin-Plus (Promega #N2512)), 15 µL Tween-20 (Sigma #P9416), 2.5 µL BSA (Roche #10735094001), 1.25 µL RNasin-Plus and 232.5 µL nuclease-free water). The samples were chopped for 5 min on ice (using scis-sors), after which the suspension was transferred to a Dounce homogenizer. Another 0.5 mL of TST-buffer was used to rinse the Eppendorf tube, which was also transferred to the Dounce ho-mogenizer. Homogenization was performed for 5 min on ice (the loose pestle was used during the first 2.5 min, the tight pestle was used during the second 2.5 min). The homogenized solution was then filtered through a 40 µm strainer and transferred to a 50 mL Falcon tube. Three addi-tional washes, using 0.5 mL of 1X ST-buffer each (1:1 of 2X ST-based buffer, in nuclease-free wa-ter), were performed to wash the Dounce homogenizer and filter. The homogenized solution was then subsequently filtered through 20 μm, 10 μm and 5 μm cell strainers placed in 50 mL Falcon tubes, washing after each filtration with 0.5 mL of 1× ST buffer, ensuring gentle pipetting to avoid formation of air bubbles. The sample was then transferred to a 15 ml Falcon tube and centrifuged at 4 °C for 5 min at 500g, followed by resuspension of the pellet in 300 μL 0.04% UltraPure BSA (Thermo Fisher Scientific). scRNAseq-3prime: autopsy samples (minced into pieces (as small as possible) upon collection) were transferred to 5 mL digestion medium (50 mL Falcon tube) consisting of Knock-OutTM Dulbecco's Modified Eagle medium (Thermo Fisher Scientific, Cat#10829018), supplemented with 1% Penicil-lin/Streptomycin (Thermo Fisher Scientific, Cat#15140122), 2X Antibiotic-Antimycotic (Thermo Fisher Scientific, Cat#15240062), 1 mM Sodium Pyruvate (Thermo Fisher Scientific, Cat#1360070),  1X DMEM NEAAs (Thermo Fisher Scientific, Cat#11140035), 1X Endothelial Cell Growth Factor supplements (ECGS/ Heparin) (PromoCell, Cat#C-30120), and containing 0.1% collagenase I, 0.1% collagenase II (Thermo Fisher Scientific), 75 µL DNase I (75 U/mL) and 1 mL Dispase. Samples were incubated for 30 min at 37°C, with pipetting (P1000) every 5 min. Next, 5 mL of cold wash buffer (PBS containing 0.5% bovine serum albumin (BSA) (Sigma-Aldrich) and 2mM EDTA) was added and the samples were filtered using a 100 µm strainer. The original Falcon tube and strainer were rinsed with an additional 3 mL of wash buffer, and the filtered suspension (appr. 13 mL) was filtered through a 40 um cell strainer into a new 50 mL Falcon tube. The sample was then transferred to a 15 mL Falcon tube, followed by centrifugation (300xG) at room temperature (RT) for 4 min and by resuspension of the pellet in 0.04% UltraPure BSA (Thermo Fisher Scientific #AM2616). Pellets were kept on ice at all times. snRNA-seq: Nuclei were counted using an automated cell counter (Luna, Logos Biosystems), followed by loading onto Chromium Chips (aimed recovery of 10,000 nuclei per sam-ple) for the Chromium Single Cell 3′ Library (V3.1) according to the manufacturer's recommenda-tions (10x Genomics). Libraries were sequenced on an Illumina NovaSeq 6000. scRNA-seq:single cell suspensions were converted to barcoded Drop-seq libraries by using the Chromium Sin-gle Cell 3' Library, Gel Bead & Multiplex Kit and Chip Kit (10X Genomics; Pleasanton, California, USA), aiming for an estimated number of 10,000 cells per library. snRNAseq: Libraries were sequenced on an Illumina NovaSeq 6000. scRNAseq-3prime: Libraries were sequenced on an Illumina NovaSeq 6000.