Illumina HiSeq 2500 paired end sequencing; mRNA-seq of the jejunum mucosa in LSL and LB laying hens following the variations of dietary calcium and phosphorus levels
mRNA-seq of the jejunum mucosa in LSL and LB laying hens following the variations of dietary calcium and phosphorus levels
The hens were slaughtered at the age of 31 weeks for tissue sample collection.  They 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  Intestinal tissue samples were ground into powder in liquid nitrogen, and approximately 50 mg of the sample was homogenized in 1 ml of TRIzol Reagent (Invitrogen, Karlsruhe, Germany). Total RNA was extracted according to the manufacturer's instructions. Extracted RNA was additionally treated with DNaseI using RNase-Free DNase Set (Qiagen) and purified using the RNeasy Mini Kit (Qiagen). The RNA quantity and quality was assessed using a NanoDrop ND-1000 (Peqlab) and a Bioanalyzer 2100 (Agilent Technologies), respectively.   2 µg of total RNA with RIN > 8 was used for library preparation using a TruSeq Stranded mRNA Kit with 11 cycles of PCR-amplification according to the manufacture's recommendation (Illumina).