title: Illumina NovaSeq 6000 sequencing; Responses to SARS-CoV-2 variants in hACE2 transgenic mice
design: Responses to SARS-CoV-2 variants in hACE2 transgenic mice
library_construction_protocol: Lung or spleen were harvested in PBS (for lung) or complete RPMI medium (cRPMI; RPMI-1640 supplemented with 1 mM L-glutamine, 0.5 mM sodium pyruvate, 10 % FCS, 100 U/ml penicillin, 0.1 mg/ml streptomycin and 25 mM HEPES) for flow cytometry, directly homogenized in PBS supplemented with protease (cOmplete™; Roche, Basel, Switzerland) and phosphatase (PhosSTOPTM; Roche, Basel, Switzerland) inhibitors for cytokine measurements or homogenized in TRIzol LS Reagent (Invitrogen, Waltham, MA, USA) for further RNA extraction. Tissue was homogenized using the gentleMACS™ Dissociator (Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany) and stored at -80 C.  RNA extraction was done on a KingFisher Flex instrument (Thermo Fisher Scientific, Germany) using the RNAdvance Tissue Kit (Beckman Coulter, Germany) and following the manufacturer's instructions. A DNA-digestion step was included in the protocol using the RNase-Free DNase Set (Qiagen, Germany). From 5 µg of the total RNA the poly(A)+ fraction was separated using the Dynabeads mRNA DIRECT Micro Kit (Invitrogen) following the manufactures instructions.  The poly(A)+ RNA was subsequently used for library preparation with the Collibri Stranded RNA Library Prep Kit for Illumina Systems (Invitrogen) following the manufactures instructions.