Transcriptomic Characterization of the Novel Avian-Origin Influenza A (H7N9) Virus: Specific Host Response and Responses Intermediate between Avian (H5N1 and H7N7) and Human (H3N2) Viruses and Implications for Treatment Options

Josset, Laurence; Zeng, Hui; Kelly, Sara M.; Tumpey, Terrence M.; Katze, Michael G.

RNA isolation and microarray processing. RNA extraction from IAV-and mock-infected Calu-3 cells in quadruplicate was performed as previously described (41) . Probe labeling and microarray slide hybridization for each biological replicate were performed using a SurePrint G3 Human Gene Expression 8Ï«60K v2 microarray (Agilent Technologies) according to the manufacturer's instructions. Slides were scanned on an Agilent DNA microarray scanner (model G2505B) using the XDR setting, and raw images were analyzed using the Agilent Feature Extraction software program (version 9.5.3.1). Extracted raw data were background corrected using the norm-exp method with an offset of 1 and quantile normalized using the limma software package (42) in the R environment. Replicated probes were mean summarized. All probes were required to pass Agilent quality control (QC) flags ("gIsFound," "gIsWellAboveBG," "gIsSaturated," "gIsFeatNonUnifOL," and "gIsFeatPopnOL") for all replicates of at least one infected time point (29, 382 probes passed QC filtering). For each sample, a log 2 FC value was calculated as the difference between log 2 -normalized data for this sample and the average of log 2normalized data for time-matched mocks. Microarray annotation was retrieved from the GEO data bank (GEO accession no. GPL17077).