Illumina HiSeq 2500 sequencing; microRNA-seq profiling of the jejunum mucosa of two laying hen strains fed different dietary Ca and P levels at the egg production peak of 31 weeks of age
microRNA-seq profiling of the jejunum mucosa of two laying hen strains fed different dietary Ca and P levels at the egg production peak of 31 weeks of age
21B11099_s
The hens were slaughtered at the age of 31 weeks for tissue sample collection. Before slaughtering, feed was deprived 2 h followed by 1 h ad libitum access to feed to standardize gut fill. The hens were individually stunned with a gas mixture of 35% CO2, 35% N2, and 30% O2 and killed by decapitation. The jejunum, terminal ileum, and ceca were identified and cut lengthwise. The digesta was gently removed and the gut was rinsed with saline. Jejunal mucosa was scraped off carefully using microscopic slides and flash frozen into liquid nitrogen, transported on dry ice to laboratory and subsequently stored at -80°C until analysis.  Birds were housed in group pens for the first 26 weeks and then rehoused into single cages. The feeding trial lasted for 21 days and the chickens were phenotyped for zootechincal and physiological parameters   Total RNA was extracted from approximately 50 mg of tissue sample using Qiazol Lysis reagent (Qiagen) and further enriched for small RNAs using a miRNeasy Mini kit (Qiagen) according to the manufracturer's recomendations. The integrity of total RNA was assessed using an Agilent RNA 6000 Nano kit and the enrichment and concentration of microRNAs were determined using an Agilent small RNA kit and the 2100 Bioanalyzer system (Agilent Technologies).   Small RNA sequencing libraries were generated from 1 µg of enriched small RNA using the NEXTFLEX Small RNA-Seq Kit v3 for Illumina according to the manufracturer's recommendations (Bioo Scientific Corp). The smRNA-seq libraries were quality assessed for expected fragment length of a major peak at about 152bp using an Agilent High Sensitivity DNA kit and 2100 Bioanalyzer (Agilent Technology) and then size-selected using the BluePippin System and 3% agarose gel cassette with the internal Q2 DNA marker (Sage Science). The molar concentration of the libraries was determined using a KAPA library quantification kit (KAPA Biosystem) and normalized to 2nM prior to sequencing.