Bioanalyzer 2

The feather follicle tissue was incubated at 4 °C overnight for penetration by RNALater solution and then transferred to −20 °C before further isolation of total RNA. Epithelium was dissected from the follicle tissue and separated from the mesenchyme in Calcium-Magnesium Free Saline (CMFS 2X) on ice (Chuong 2000 ). Total RNA from feather epithelium was insolated using the RNEasy Plus Mini Kit (Qiagen, Hilden, Germany) with an additional on-column DNase treatment recommended by the manufacture (Qiagen). The 15-min DNase treatment was carried out at room temperature by mixing 10 μl DNase and 70 μl RDD buffer and applied to the RNA-binding column after the first wash. The RNA quantities and qualities of each individual were analyzed by NanoDrop (Thermo Scientific, Waltham, MA) and BioAnalyzer II (Agilent Technologies). If all samples from the same litter passed the quality control (RNA integrity number > 8.0), 10 µg of total RNA from each sample would be pooled to reach a final of 30 µg total RNA for sequencing for each sample.

The total RNA from about 50 mg of colon tissues was extracted using TRIzol reagent, and then the purity, concentration, and integrity of the total RNA were analyzed using NanoDrop and BioAnalyzer II reagents (Agilent, CA, USA). Next, the RNA-Seq library was established for mRNA sequencing. The sequencing was conducted using an Illumina HiSeq 4000 at the LC-BIO (Hangzhou, China). Gene expression levels were assessed by fragments per kilobase of transcript per million fragments mapped (FPKM). The differentially expressed genes (DEGs) were defined by fold change > 2 and raw data p < 0.05. Database functionality annotations for DEGs were performed by Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis.