GSM9081363: hCOs, bOH2024-inoculated, 1 day post-inoculation (contemporary HPAI H5N1); Homo sapiens; Influenza A virus; RNA-Seq
Human cerebral organoids (hCOs) were dissociated into a single cell suspension as follows: one organoid per well was placed in a 24-well plate. hCOs were washed with Dulbecco's Modified Eagle Medium/F-12 (Gibco). hCOs were incubated with 500 µl trypsin EDTA (Gibco), 500µl Accutase (MilliporeSigma), and 200 units of RNase inhibitor (Applied Biosystems) for 20 minutes at 37°C and 5% CO2. Every ten minutes, hCOs were gently agitated using a wide-bore P1000 pipette tip. If hCOs were not dissociated after twenty minutes, they were incubated for a further 5 minutes. Cells were filtered through a pre-wet 70 µm filter (pluriSelect) into stop solution (PBS + 20% FBS) and centrifuged at 200 x g for 5 minutes at 4°C. The cell pellet was resuspended in Cell Suspension Buffer (Fluent Biosciences), and filtered through a pre-wet 40 µm filter (pluriSelect). Cell concentration was determined using trypan blue and a manual hemocytometer. The recommended 5,000 cell input was used for the PIPseq T2 protocol (Fluent Biosciences). Cells derived from 3 organoids per sample were pooled for scRNA-seq library construction. Libraries were prepared using PIPseq T2 3' Single Cell RNA kit v4.0 PLUS (Fluent Biosciences) according to the manufacturer's instructions. Samples were indexed using the PIPseq UDI-96 kit (Fluent Biosciences)