title: GSM8505476: T1DM infected with MA-SARS-CoV-2; Mus musculus; RNA-Seq
library_construction_protocol: Given the requirements for ABSL-3 safety precautions, the tissue harvested from these mice needs to be fixed in paraformaldehyde for at least 24 hours prior to further handling. Logistically, the digestion of tissue into a single cell suspension prior to the required fixation step was impossible due to time and staff constraints. To facilitate these experiments, we adapted a novel technique originally developed for the analysis of fixed tissue for scRNA-seq, Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling (10x Genomics, Protocol CG000553). Tissue was harvested in the ABSL-3 facility by trained personnel and minced into square millimeter sized pieces before being fixed for 24 hours at 4ºC. Samples dedicated to scRNA sequencing were fixed with Fix & Perm Buffer (10x Genomics, PN-2000517) as per manufacturer's protocol while samples for flow cytometry were fixed in 4% PFA. Samples were then washed with PBS and the fixation reaction was quenched. scRNA samples were quenched with Quench Buffer (10x Genomics, PN-2000516) as per manufacturer's protocol, while flow cytometry samples were quenched in solution of 50mM glycine dissolved in PBS. Tissue was then dissociated into a single cell suspension shaking vigorously at 37ºC for 1 hour in Dissociation Solution made with LiberaseTM TL (concentration 0.2mg/mL, Millipore Sigma, PN 5401020001) as per manufacturer's protocol. Tissue was further triturated with a P1000 pipette tip 15-20 times before being filtered through a 40µM filter, which was then washed with PBS. The samples were quenched again with Quench Buffer and cells were then counted by hemocytometer before proceeding to scRNA sequencing or flow cytometry. Samples were stored at 4ºC for up to 1 week before use. Samples designated for scRNA sequencing were incubated in Quenching Buffer and Enhancer (10x Genomics, PN- 2000482) as per manufacturer's protocol. Single-cell sequencing libraries were created according to 10x Genomics Chromium Fixed RNA Profiling Reagents (10x Genomics, PN 1000414) and associated user protocol (User Guide CG000477 Rev D). After fixation, the samples were first hybridized using mouse specific whole transcriptome probe pairs. Cell counts were verified using a LogosBio Luna-FL automated cell counter(1.05e6 cells•mL-1and 2.8e6 cells•mL-1for sample 708L and 719L, respectively). The cells were pelletized and re-suspended in a hybridization mixture prepared using 10x Genomics Hyb Buffer B (PN 2000483) and Enhancer (PN 2000482) according to the protocol. Mouse specific WTA Probes BC001 (10x Genomics, PN 2000703) were then added to each sample at room temperature. The samples were incubated at 42°C in a Bio-Rad C1000 Touch Thermal Cycler for 16 hours.