RNA-seq of medullary bone of two high-yielding laying hen strains during the transition from immature pullet stage to the onset of laying
RNA-seq of medullary bone of two high-yielding laying hen strains during the transition from immature pullet stage to the onset of laying
The animal trial was performed at the Agricultural Experimental Station of the University of Hohenheim, Germany. The experimental protocol was in strict compliance with the German Animal Welfare Legislation and approved by regional Ethics committee, i.e., the Regierungspräsidium Tubingen, Germany, and the Animal Welfare Committee of the University of Hohenheim (Project no. HOH67-21TE). Hens were fed with experimental maize-soybean meal-based diets containing all required nutrients according to their developmental stages from developer feed (week 14-15) to pre-layer feed (week 15-16) and finally to layer feed (week 16-24). The hens were terminated for sample collection at 16, 17, 18, 20, 21, 22 and 23 weeks of age. Femurs of the left leg were cut at the metaphysis, medullary bone material was collected, and frozen at -80 °C until analysis.  Frozen medullary bone was ground with pestle and mortar in liquid nitrogen. Total RNA was isolated using TRI reagent (Sigma-Aldrich) and phenol-chloroform-based extraction according to the TRI reagent protocol. To remove trace carry-over DNA contamination, the RNA was treated with DNase I (Roche Diagnostics) and purified using the NucleoSpin RNA extraction kit (Macherey-Nagel). The RNA concentration was determined using the NanoDrop ND-2000 (Peqlab) and the RNA quality was analyzed using Bioanalyzer 2100 device (Agilent Technologies). High quality RNA (RIN > 7) was used for sequencing library construction using the Illumina Stranded mRNA preparation kit (Illumina, San Diego) according to the manufacturer's recommendations.