title: scRNA-seq of homo sapiens: SARS-CoV-2 infected lung tissue
design: BSL2: Cells isolated from normal lung tissue were captured in droplet emulsions using a 10x Chromium Controller (10x Genomics). cDNA was recovered and libraries were prepared using 10x Genomics 3 or 10x Genomics 5 Single Cell V3.1 protocol (infections 1,2,4 and 5 were sequenced using 3 chemistry, while infection 3 used both 3 and 5 technology), as described32. Sequencing libraries were analyzed (Agilent TapeStation D4150, using regular and high sensitivity D5000 ScreenTape) and balanced, and sequenced on a NovaSeq 6000 (Illumina).  BSL3: The 10x Genomics Single Cell protocols were performed as before, with the following modifications for BSL3. The 10x Genomics, 3 or 5 Single Cell v3.1 master mix was prepared outside the BSC. Within the BSC, cells prepared as above were added to the master mix in PCR tubes (USA Scientific 1402-4708) in a 96-well cold block (Ergo 4ti-0396) and the 10x chip was loaded per manufacturers instructions, sealed, and processed in a 10x Chromium Controller in the BSC. The resultant cell/bead emulsions were loaded into PCR tubes and transferred immediately to a pre-warmed (53) PCR machine for cDNA synthesis carried out at 53 for 45 minutes, then 85 for 5 minutes, then 60 for 15 minutes (plaque assays showed that exposure of SARS-CoV-2-infected samples at 60 for 20 minutes in this manner rendered the sample non-infectious). After cDNA synthesis, samples were transferred out of the BSL3 for cDNA recovery, amplification, and sequencing library preparation as above.