title: GSM4832787: covid-19_study1_pt3; Homo sapiens; RNA-Seq
library_construction_protocol: bulk RNA-seq: RNA was isolated from snap frozen tissue after homogenization in TRIzol using a tissue homogenizer (Precellis 24 Homogenizer, Bertin Technologies or Ribolyser, MP Biomedicals) using standard procedures. For RNA preparation from formalin fixed paraffin embedded (FFPE) material, up to 5 10-um tissue sections were pooled, deparaffinized with Deparaffinization Solution (Qiagen) and RNA was isolated using the miRNEASY FFPE kit (Qiagen) according to the manufacturer's instructions. scRNA-seq: Autopsy samples were rinsed with PBS, minced into pieces (as small as possible), and transferred to a low-bind Eppendorf containing 2 mL digestion medium consisting of Dulbecco's Modified Eagle medium (DMEM) supplemented with 2mg/mL (100X stock: 200 mg/mL) Collagenase P (Roche) and 0.2 mg/mL (100x Stock: 20 mg/mL) DNAse I (Roche). Samples were incubated for 15 min at 37°C, cells were pipetted every 5 min to rupture the larger pieces. Next, cells were filtered using a 40 µm strainer, followed by addition of 2 mL cold PBS to wash the sample. The suspension was then transferred to several Eppendorf tubes (on ice), followed by centrifugation at 300xG (4°C) for 5 minutes. Supernatant was discarded, and the cells were resuspended with RBC lysis buffer (total 250-500 µL) and combined into one vial. After 5 min of incubation, the suspension was centrifuged at 200xG (4°C) for 5 minutes. Supernatant was discarded as much as possible, and samples were resuspended in 100 µl PBS containing 0.04% UltraPure BSA (Thermo Fisher Scientific) and filtered over a 40µm tipstrainer on ice. The number of cells and fraction of live cells in the suspension was counted using an automated cell counter (Luna, Logos Biosystems). Throughout the dissociation procedure, cells were maintained on ice whenever possible. bulk RNA-seq (human): Sequencing libraries were  prepared with the Lexogen QuantSeq 3' mRNA-Seq library prep kit  according to the manufacturer's instructions. Samples were indexed to allow  for multiplexing. Library quality and size range was assessed using a  Bioanalyzer (Agilent Technologies) with the DNA 1000 kit (Agilent  Technologies, California, USA). Libraries were diluted to a final concentration of 2nM and subsequently sequenced on an Illumina HiSeq4000 instrument. bulk RNA-seq (hamster): Sequencing libraries were prepared using non-stranded & polyA-selected RNA preparation. scRNA-seq: Single cell suspensions were converted to barcoded Drop-seq libraries by using the Chromium Single Cell 5' Library, Gel Bead & Multiplex Kit and Chip Kit (10X Genomics; Pleasanton, California, USA), aiming for an estimated number of 5,000 cells per library. bulk RNA-seq (human): Single-end reads of 50 bp length were produced with a minimum of 1M reads per sample. bulk RNA-seq (hamster): Pair-end sequencing (100 cycles (PE100)) was performed using DNBSED technology and a MGI2000 sequencer. scRNA-seq: Libraries were sequenced on an Illumina NovaSeq 6000.