Ultra 2 directional rna library kit

Three to four independent biological replicates (5 flies per group) of normoxia- and hypoxia-exposed groups of w¹¹¹⁸ and foxo mutants were prepared and analyzed. RNA-sequencing was conducted by the University of Calgary Centre for Health Genomics and Informatics. The RNA Integrity Number (RIN) was determined for each RNA sample. Samples with an RIN score higher than 8 were considered good quality, and Poly-A mRNA-seq libraries from such samples were prepared using the Ultra II Directional RNA Library kit (New England BioLabs) according to the manufacturer’s instructions. Libraries were then quantified using the Kapa qPCR Library Quantitation kit (Roche) according to the manufacturer’s directions. Finally, RNA libraries were sequenced for 100 cycles using the NextSeq 500 Sequencing System (Illumina). Transcripts were quantified using kallisto (Bray et al. 2016 (link)) referencing refSeq mRNA (release: October 15, 2019) corresponding to dm6 annotation. Differential expression testing was performed using sleuth (Pimentel et al. 2017 (link)).

Six independent biological replicates (5 flies per group) of mock infected and P.e. infected groups were prepared and analyzed. RNA-sequencing was conducted by the University of Calgary Centre for Health Genomics and Informatics. The RNA Integrity Number (RIN) was determined for each RNA sample (6 replicates per each condition were used). Samples with a RIN score higher than 8 were considered good quality, and Poly-A mRNA-seq libraries from such samples were prepared using the Ultra II Directional RNA Library kit (New England BioLabs) according to the manufacturer’s instructions. Libraries were then quantified using the Kapa qPCR Library Quantitation kit (Roche) according to the manufacturer’s directions. Finally, RNA libraries were sequenced for 100 cycles using the NextSeq 500 Sequencing System (Illumina).