02% Coomassie blue G-250, and the anode buffer contained 25 mM imidazole. Proteins were separated at 12 milli-amps for 2 hours in 4°C. Immunoblot analyses PAGE separated proteins were transferred to PVDF using tank transfer at 350 milliamps for 1 hour, blocked with 5% milk for one hour and probed with anti-Ago2 Ab diluted 1:100 [3]. ECL Plus chemiluminescence detection was used, and the blot was exposed to ECL film (Amersham). Acknowledgements We thank the Arthropod-borne click here and Infectious
Diseases Lab Core Support for providing mosquitoes and viral titrations. We are also grateful to Richard Casey of the Bioinformatics Center of Colorado State University for providing support during preliminary investigations of analytical methods. This work
was funded by the SOLiD™ System $10 K Genome Grant Program sponsored by Life Technologies (CLC, AP), Gates Foundation/NIH Foundation grant (CLC, KEO), and by funds from the National Institute of Allergy and Infectious Disease, National Institutes of Health, under grant AI067380 (GDE, ANP). Electronic supplementary material Additional file 1: Additional viRNA profiles. A. sRNA reads from representative libraries of un-infected controls show non-specific alignment to the DENV2 genome. Panels from left to right indicate, 2, 4, and 9 dpi, respectively. Top panel shows count distribution along DENV2 genome for a representative library AZD6738 at each timepoint. Bottom panel shows mean sRNA distribution by size. Blue and red bars indicate sense and anti-sense sRNAs, respectively. B. viRNA WebLogos. viRNAs from a representative 9 dpi DENV2-infected cohort were separated by size group and subjected to WebLogo sequence alignment http://weblogo.berkeley.edu/ to identify the relative nucleotide frequency at each position. About Niclosamide 20,000 reads were analyzed for the combined categories. C. 24-30 nt piRNAs are more
abundant in DENV2-infected samples. Total mean transcriptome-mapped reads of un-infected and DENV2-infected libraries categorized by sRNA size group. Blue and red bars indicate sense and anti-sense viRNAs, respectively. (PDF 108 KB) Additional file 2: Host sRNA Profile Summary Tables. Summary data categorized by mapped read orientation and sRNA size group. ‘Summary’ page shows total sRNA reads in pooled libraries for each condition tested. ”Transcripts’ shows the number of targets remaining after removing low-abundance (<10 reads) and flagged candidates. “”Flagged”" segments are those for which a replicate accounted for 70% or more of the total reads; these were deleted from the final analysis. ‘Enriched’ and ‘Depleted’ indicate the number of targets showing significant changes in DENV2-infected pools over controls. Significance was determined using the edgeR exact test, and a Benjamini-Hochberg cut-off of 0.05 was used to adjust for multiple testing and control the false discovery rate. The following pages list raw sRNA count data for each target transcript at 2, 4, or 9 dpi.