RNA was isolated from snap frozen kidneys by homogenization in Trizol using a Retsch MM300 Tissue Lyser. Chloroform was added and the aqueous phase was processed using the Agencourt RNAdvanced tissue kit that was modified for automation on a Biomek FXp from Beckman. For Taqman analysis, reverse transcription was performed using the TaqMan Reverse Transcription Reagents Kit (Applied Biosystems). The resultant cDNA was used in a ViiA 7 Real-Time PCR system (Applied Biosystems) using mouse specific probes from Applied Biosystems. Statistical analysis was done by ANOVA, followed by Dennett’s T-test in GraphPad Prism.
RNA seq was performed commercially by Labcorp (Seattle, WA). Single end RNA-seq reads were mapped with STAR 2.4. Absolute read counts/gene were generated using Subread’s feature Counts program v1.4.6 and the gtf annotation file for mouse mm10 reference from UCSC. Differential expression analysis was performed with the DESeq2 package. Functional annotation analysis for the differentially expressed genes was performed using pathway analysis tools including Ingenuity Pathway Analysis (QIAGEN) and MetaBase (Thomson Reuters).
RNA seq was performed commercially by Labcorp (Seattle, WA). Single end RNA-seq reads were mapped with STAR 2.4. Absolute read counts/gene were generated using Subread’s feature Counts program v1.4.6 and the gtf annotation file for mouse mm10 reference from UCSC. Differential expression analysis was performed with the DESeq2 package. Functional annotation analysis for the differentially expressed genes was performed using pathway analysis tools including Ingenuity Pathway Analysis (QIAGEN) and MetaBase (Thomson Reuters).