RNA-seq reveals altered gene expression levels in proximal tubular cell cultures compared to renal cortex but not during early glucotoxicity
Cite this dataset
Brismar, Hjalmar; Nilsson, Linnea; Scott, Lena; Castresana Aguirre, Miguel (2020). RNA-seq reveals altered gene expression levels in proximal tubular cell cultures compared to renal cortex but not during early glucotoxicity [Dataset]. Dryad. https://doi.org/10.5061/dryad.v9s4mw6rb
Abstract
Methods
Cell culture and tissue preparation
Twenty-day-old male Sprague Dawley rats were used for preparation of proximal tubule slices and PTC cultures. All animals were housed under controlled conditions of light and dark (12:12 hours) and given a standard diet containing 20 % protein by weight and tap water were available ad libitum. All experiments were performed according to Karolinska Institutet regulations concerning care and use of laboratory animals and were approved by the Stockholm North ethical evaluation board for animal research.
Proximal tubule slices were collected from the outer 150 µm of the renal cortex, where 90 % of the tubular volume is proximal tubules (3). Primary cultures of rat PTC were prepared as previously described (1) using the outer 150 µm renal cortex as starting material. Cells were seeded in 60-mm wells and cultured in 37°C at an approximate humidity of 95-98 % with 5 % CO2 for three days before experiments. Culture medium was changed every 24 hours. Cells were exposed to 15 mM glucose (HG) or 5.6 mM glucose (control) for 8 hours. Kidney cortex samples were prepared in replicates from three animals. PTC samples were prepared from three separate cultures and pairwise exposed to HG or control.
RNA-seq
Cells and tissue samples were collected and mRNA extracted and purified with RNeasy mini kit (cat. no 74134, Qiagen AB, Sollentuna, Sweden) following manufacturer’s instructions. The quality of the starting RNA was validated with an Agilent Bioanalyzer before cDNA libraries were created. The cDNA libraries were created by National Genomics Infrastructure at Science for Life Laboratory (Solna, Sweden) using Illumina TruSeq Stranded mRNA with poly-A selection. Each sample was used to generate two separate cDNA libraries. Quality controls of the libraries were performed by National Genomics Infrastructure at Science for Life Laboratory (Solna, Sweden) using MultiQC.
Usage notes
The data is stored as tar archives. Each tar file contains FASTQ RNAseq data organised according to treatment
P10265_101 renal cortex
P10265_102 renal cortex
P10265_103 renal cortex
P10265_105 PTC cultured 3 days
P10265_106 PTC cultured 3 days
P10265_202 PTC cultured 3 days
P10265_108 PTC cultured 3 days High Glucose
P10265_109 PTC cultured 3 days High Glucose
P10265_203 PTC cultured 3 days High Glucose
Funding
Swedish Research Council
Science for Life Laboratory
Knut and Alice Wallenberg Foundation
Märta och Gunnar V. Philipsons Stiftelse
Märta and Gunnar V Philipson foundation