Project description:Acute kidney injury (AKI) is an important contributor to the development of chronic kidney disease (CKD). We performed miRNA and mRNA sequencing on biobanked human kidney tissues obtained in the routine clinical care of patients with the diagnoses of AKI and minimal change disease (MCD), in addition to nephrectomized (Ref) tissue from individuals without known kidney disease. From all renal biopsy samples, 2 cryosections (10 μM) including the entire cross-section of the tissue were placed directly into PicoPure RNA extraction buffer. RNA was isolated using the PicoPure isolation kit. Total RNA was evaluated for quantity and quality using an Agilent Bioanalyzer. Approximately 100 ng of total RNA was used for library construction with QIASeq miRNA Library Kit. Each resulting indexed library was quantified and its quality assessed by Qubit and Agilent Bioanalyzer. The libraries were then loaded onto an Illumina NextSeq 500 for 75 bp single-read sequencing to a depth of 15-20M reads per library. Sequence reads were uploaded to the Qiagen GeneGlobe Data Analysis Center (https://www.qiagen.com/us/resources/geneglobe) for quality control, alignment and expression quantification. Reads were mapped to different databases for mature, hairpin, noncoding RNA, mRNA and other RNA using bowtie v1.2. Read counts and UMI counts for each RNA category (mature, hairpin, piRNA, tRNA, rRNA, mRNA and otherRNA) were calculated for each sample, using miRbase V21 for miRNA, and piRNABank for piRNA.
Project description:Acute kidney injury (AKI) is an important contributor to the development of chronic kidney disease (CKD). We performed miRNA and mRNA sequencing on biobanked human kidney tissues obtained in the routine clinical care of patients with the diagnoses of AKI and minimal change disease (MCD), in addition to nephrectomized (Ref) tissue from individuals without known kidney disease. From all renal biopsy samples, 2 cryosections (10 μM) including the entire cross-section of the tissue were placed directly into PicoPure RNA extraction buffer. RNA was isolated using the PicoPure isolation kit. Total RNA was evaluated for quantity and quality using an Agilent Bioanalyzer. Approximately 10 ng of total RNA were used for each sample’s library preparation. Ribosomal RNA was removed using RiboGone – Mammalian Kit protocol. After rRNA depletion, double-stranded cDNA was synthesized and amplified using the SMARTer Universal Low Input RNA Kit protocol. The cDNA was sheared by Covaris, and the library was prepared and barcoded following the Ion Plus Fragment Library Kit protocol. Each resulting barcoded library was quantified and quality assessed by Agilent Bioanalyzer. Multiple libraries were pooled in equal molarity. Finally, 8 μL of 100 pM pooled libraries were loaded into lanes of an Illumina HiSeq 4000. At least 30M reads per library were generated.
