Illumina HiSeq 2500 paired end sequencing; RNA-seq analysis of kidney and jejunum of German Landrace sows fed high, medium and low level of dietary phosphorus
Sows were slaughtered at day 29 of lactation (corresponds to day 154 on trial) and individual tissue samples were taken. In brief, the left kidney was cut open and cortex was retrieved from the lateral part. For the intestine, 10 cm of jejunum was dissected at a distance of 200 cm from the stomach. The intestinal tube was cut open to remove digesta and rinse the surface with ice-cold NaCl (0.9%) to remove residual contaminants. The intestinal epithelium was scraped off with a scalpel. All samples were snap frozen in liquid nitrogen immediately after preparation and stored at -80 °C until RNA isolation. Total RNAs were isolated from all tissue samples using TRI Reagent according to user guides (Sigma-Aldrich, Taufkirchen, Germany), followed by Baseline-ZERO DNase treatment (Biozym, Hessisch Oldendorf, Germany) and purification with the column-based NucleoSpin RNA II-Kit (Macherey–Nagel, Düren, Germany). Concentration of purified RNA samples was measured by Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, USA). The quality of RNA extracts were checked on a Bioanalyzer 2100 (Agilent Technologies, Waldbronn, Germany) yielding RNA integrity numbers (RIN) from 6.3 to 9.1 (average of 8.0).  RNA libraries were prepared from the final purified total RNAs according to the TruSeq Stranded mRNA protocol (Illumina, San Diego, CA, United States) and the quality was validated using a Agilent DNA-1000 chip kit (Bioanalyzer 2100).