DNA methylation profiling of the jejunum mucosa of two laying hen strains (Lohmann Brown-Classic (LB) and Lohmann LSL-Classic (LSL)) at 5 different ages
DNA methylation profiling of the jejunum mucosa of two laying hen strains (Lohmann Brown-Classic (LB) and Lohmann LSL-Classic (LSL)) at 5 different ages
Tissue samples were dissected from the animals following an anatomical reference guide. 2 cm long jejunum sample was taken about 3 cm distal to the duodenal loop of each bird, cut open and rinsed with sterilized saline buffer to remove digesta residues. The intestinal epithelium was scraped using sterilized razor blades. The mucosa sample was frozen on dry ice and stored at -80°C upon DNA extraction. All animal care and sample collection procedures were in compliance with the German Animal Protection Law and approved by the FBN animal care committee.  Genomic DNA was extracted from samples using a DNeasy kit (Qiagen) according to the manufacturer's recommendation including proteinase K digestion and RNase A treatment to obtain high purity DNA.  2 µg of DNA was spiked-in with 1% DNA control of unmethylated cl857 Sam7 Lambda DNA (Promega) and digested for 7 hours with 100U MspI (New England Biolabs) and then overnight with 50U TaqαI. The DNA fragments (double-enzyme digested) were end-repaired, A-tailed and ligated to TruSeq DNA adapters using a TruSeq Nano DNA library prep kit (Illumina) according to the manufacturer's protocols. Size-selection of the adapter-ligated DNA fragments were achieved by using sample purification beads (TruSeq Nano DNA library prep kit) to recover DNA inserts around 40–240bp. The adapter-ligated DNA libraries was subjected to a bisulfite conversion using an EZ DNA Methylation-Gold Kit according to the manufacturer's protocols (Zymo Research). Library amplification was performed using a PfuTurbo Cx Hotstart DNA Polymerase kit (Stratagene). The quality of libraries was assessed on 2100 Bioanalyzer using a high-sensitive DNA chip (Agilent Technologies).