GSM9409158: kidney, BS, RNA 1257; Rattus norvegicus; RNA-Seq
RNA was obtained from frozen tissue blocks using Trizol Reagent (Invitrogen, Thermo Fisher). Genomic DNA was removed by incubation with DNase I for 10 minutes (Invitrogen) and QC was assessed in a TapeStation 2200 (Agilent, Santa Clara, USA). Half of RNA were used for RNA-Seq experiments and the rest were used for qPCR purposes. SmallRNAs from tissue were purified using 1mg of total isolated RNA following an enrichment protocol with SPRIselect beads (Bee Na Lee, Beckman Coulter, California, USA). qPCR validations were done . The urine samples were collected and 500ml (total cell-free and exosomal RNA) used for smallRNA obtention following the instructions of the ZR Urine RNA Isolation Kit (Zymo Research Corp, CA, USA). In both cases, assessment of QC was done in a Bioanalyzer using a High Sensitivity chip (Agilent). RNA-seq Libraries were prepared from 1mg of total isolated RNA with the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, NEB, Massachusetts, USA), run in a HighSeq 2500 System (Illumina, CA, USA) and 40-50M of reads per sample were obtained. The quality of reads was assessed using the FastQC tool and a trimming step was applied to remove the adapters using the Cutadapt tool. Salmon tool was used to map and quantify the aligned transcripts to the Rnor6.0 genome. DESeq2 v1.24.0 Bioconductor's package has been used for differential expression testing and the analysis of biological significance has been based on Gene Set Enrichment Analysis (GSEA) performed over two annotation databases: Gene Ontology (GO) and the Reactome Pathway Knowledge base. smallRNA libraries were prepared following the instructions of the NebNext Small RNA Library Prep Set for Illumina (NEB). Size selection of fragments was done in a 6% Polyacrylamide Gel (Invitrogen) and QC of libraries in a TapeStation 2200 using a HighSens D1000 screentape (Agilent). Libraries were sequenced in a HighSeq 2500 System (Illumina) and 12-15M reads per sample were obtained and analyzed. The quality of reads was assessed using the FastQC tool and a trimming step was applied to remove the adapters using the Cutadapt tool. The Subread/Rsubread package was used to map and quantify the aligned transcripts to the Rnor6.0 genome. For summarization/quantification step, annotations were obtained from different databases depending on the small RNA class: miRNA precursor and mature annotation was derived from miRBase v22 (http://www.mirbase.org/ftp.shtml), whereas annotations for other non-coding smallRNAs described in rat were derived from Ensembl release 102 (https: //www.ensembl.org/info/data/ftp/index.html). smallRNA libraries were prepared following the instructions of the NebNext Small RNA Library Prep Set for Illumina (NEB). Size selection of fragments was done in a 6% Polyacrylamide Gel (Invitrogen) and QC of libraries in a TapeStation 2200 using a HighSens D1000 screentape (Agilent). Libraries were sequenced in a HighSeq 2500 System (Illumina) and 12-15M reads per sample were obtained and analyzed. The quality of reads was assessed using the FastQC tool and a trimming step was applied to remove the adapters using the Cutadapt tool. The Subread/Rsubread package was used to map and quantify the aligned transcripts to the Rnor6.0 genome. For summarization/quantification step, annotations were obtained from different databases depending on the small RNA class: miRNA precursor and mature annotation was derived from miRBase v22 (http://www.mirbase.org/ftp.shtml), whereas annotations for other non-coding smallRNAs described in rat were derived from Ensembl release 102 (https: //www.ensembl.org/info/data/ftp/index.html). Different types of quality controls were also conducted (multi-dimensional scaling plot analysis, Euclidean distances between samples) to ensure the normalized data are appropriate for the differential expression analysis; as a result, no outliers or batch effects were detected. DESeq2 v1.24.0, a Bioconductor package, was utilized for the differential expression testing. miRNA target sites were cataloged in databases based on experimental validation and computational prediction using the multiMiR Bioconductor package (http://multimir.ucdenver.edu). DESeq2 v1.24.0 Bioconductor's package was used for differential expression testing. miRNA target sites have been catalogued in databases based on experimental validation and computational prediction using the multiMiR Bioconductor's package (http://multimir.ucdenver.edu).