E z n a viral rna extraction kit

RNA was isolated from egg allantoic fluid using the E.Z.N.A. viral RNA extraction kit (Omega Bio-Tek) according to the manufacturer’s instructions and then underwent next-generation sequencing. Sequences were assembled using a pipeline designed at the Icahn School of Medicine at Mount Sinai as described previously (42 (link)). To identify point mutations, full-length coding sequences were compared to the sequenced wild-type A/Netherlands/602/2009 (H1N1)pdm09 strain used for escape mutagenesis. All reported mutations were numbered from the methionine of each protein.

Viral RNA was extracted from apical washes using the E.Z.N.A. viral RNA extraction kit (R6874-02; Omega Bio-tek, Inc., Norcross, GA, USA) and from tissue lysate using the E.Z.N.A. total RNA extraction kit (R6834-02; Omega Bio-tek, Inc., Norcross, GA, USA). For RNA quantification, primers and probes specific for each virus were used as previously described^{20 (link)}. The RNAseP housekeeping gene was used as an internal control for cell-associated virus quantification (4331182; Life Technology, Zug, Switzerland).
Quantitative real-time reverse transcriptase polymerase chain reaction (RT-qPCR) assays were performed using the QuantiTect probe RT-qPCR kit (no. 204443; Qiagen, Valencia, CA, USA) in a StepOne Applied Biosystems thermocycler (Thermo Fisher Scientific, Waltham, MA, USA). In each run and for each virus, four 10-fold serial dilutions of RNA reference standards were included. Results were analyzed using the StepOne version 2.0 software (Thermo Fisher Scientific, Waltham, MA, USA).

RNA was extracted from virus containing allantoic fluid using an E.Z.N.A. viral RNA extraction kit (Omega Bio-Tek) according to the manufacturer’s instructions and stored at −80°C for future use. Next generation sequencing was performed using a MiSeq v2, 300 cycle reagent kit (Illumina). A customized pipeline that has been implemented at the Icahn School of Medicine at Mount Sinai was used for genome assembly (50 (link)). The assembled segments were aligned using MUSCLE in MEGA 7.0 (51 (link)) to the wild-type pdmH1N1 deep-sequenced virus used at the beginning of the escape passaging. Of note, the pdmH1N1 virus used in this study has two HA mutations, N173D and Q240R, compared to the sequence that can be found on the Global Initiative on Sharing All Influenza Data (GISAID) database. The CR9114 L335V/D363G/A388T EMV HA mutations were found using Sanger sequencing through Genewiz.