Illumina NovaSeq 6000 paired end sequencing; Single-cell RNAseq of mouse brain infected with Langat virus in WT and Ifnar KO
Single-cell RNAseq of mouse brain infected with Langat virus in WT and Ifnar KO
For the experiments, animals (7- to 13-week-old, mixed gender) were either left untreated mock treated (PBS) or infected with LGTV. After sedation with ketamine (100 ug/g body weight) and xylazine (5 ug/g body weight) or Isoflurane, animals were intracranially inoculated with indicated dose of LGTV or LGTV-GFP suspended in 20 uL PBS. After euthanasia (day 5 for WT and day 3 for Ifnar-/-), brains were collected in cold Hibernate A GlutaMAX medium (Thermo Fisher) without cardiac perfusion. Single nuclei were isolated from the cerebral cortex of WT and Ifnar-/- mice using the protocol modified from Gaublomme et al 2019 (PMID: 31266958). Cerebral cortexes from the hemisphere that did not receive needle injection from one male and one female mouse were manually dissected without cardiac perfusion and homogenized in lysis buffer (10 mM Tris pH 7.4, 146 mM NaCl, 1 mM CaCl2, 21 mM MaCl2, 0.1% NP40 and 40 U/ml RNase inhibitor) using Dounce homogenizer for 10-20 times. Brain homogenates were washed with wash buffer (10mM Tris pH 7.4, 146 mM NaCl, 1mM CaCl2, 21 mM MaCl2, 0.01% BSA and 40 U/ml RNase inhibitor) and filter through 20 µm pre-separation filters (Miltenyi Biotec, 130-041-407). The homogenates were centrifuged with a swing bucket at 500xg, 4oC for 5 min. The supernatant was removed, stain buffer (10 mM Tris pH 7.4, 146 mM NaCl, 1 mM CaCl2, 21 mM MaCl2, 2% BSA and 40 U/ml RNase inhibitor) was added. The resuspended cells were filtered again and the homogenates from one male and one female were mixed. Nuclei were counted by hemocytometer and diluted in the staining buffer to 2 x 106 nuclei/ml. Nuclei were stained with 10 µg/ml DAPI and subjected to sorting from cell debris using FACS (BD FACSAria III). The gate was set to sort for DAPI positive population (FSC 302, SSC 313 and BV421 313(log). The flow rate was adjusted to 2-6 to maintain about 10 nl/nuclei and with 80% efficiency. Sorted nuclei were transferred into low binding tubes and centrifuged using a swing bucket at 500xg, 4oC for 5 min. The overall quality of sorted nuclei was assessed under a microscope. Overall, 80% viable nuclei were then diluted for single cell RNAseq processing, using 10x Genomics 3' v2 kit according to manufacturer's instructions. 10x Genomics 3' v2 kit according to manufacturer's instructions 10x Genomics 3' v2 kit according to manufacturer's instructions