Bgiseq500rs

Conjunctival epithelium was excised from B6 and Pinkie strains and total RNA was extracted using a QIAGEN RNeasy Plus Micro RNA isolation kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The concentration and purity of RNA were assessed using a NanoDrop 1000 (ThermoFisher Scientific, Waltham, MA, USA). RNA-Seq was performed by the Beijing Genomics Institute (BGI) using the BGISEQ500RS to generate 100 bp paired-end reads. The raw data were cleaned by removing reads containing adapter or poly-N sequences, and reads of low quality using SOAPnuke (v. 1.5.2, parameters: −l 15 −q 0.2 −n 0.05), and the expression levels of the resulting genes and transcripts were determined using RSEM (v. 2.2.5, default parameters). Detection of DEGs (differentially expressed genes) was performed with DEseq2 (parameters: fold change ≥ 2.00 and adjusted p-value ≤ 0.05). A total of 19,511 genes were obtained as raw data. Genes were passed through the Benjamini–Hochberg procedure to obtain the critical value for false discovery and a total of 1375 genes passed with a p-value > 0.0006. The selected genes were clustered in a heat map based on GSCA pathways.

Conjunctival epithelium was excised from B6 and Pinkie strains and total RNA was extracted using a QIAGEN RNeasy Plus Micro RNA isolation kit (Qiagen) according to the manufacturer's instructions. The concentration and purity of RNA was assessed using a NanoDrop 1,000 (ThermoFisher Scientific, Waltham, MA). RNA-Seq was performed by the Beijing Genomics Institute (BGI) using the BGISEQ500RS to generate 100-bp paired-end reads. The sequencing reads were cleaned by removing reads containing adapter or poly-N sequences, and reads of low quality using SOAPnuke (version 1.5.2, parameters: -l 15 -q 0.2 -n 0.05). and the expression levels of the resulting genes and transcripts were determined using RSEM (version 2.2.5, default parameters). A total of 19,511 genes were obtained as raw data. Detection of DEGs (differentially expressed genes) was performed with DEseq2 (Parameters: Fold Change >2.00 and adjusted P < 0.05). Genes were passed through the Benjamini-Hochberg procedure to obtain the critical value for false discovery and a total of 1,375 genes passed with a P >0.0006. The selected genes in the IL-17 signaling pathway were clustered in a heat map using GraphPad Prism 9.0 software (San Diego, CA, USA).

Total RNA was extracted from 35 human liver tissues according to the following procedures. Approximately 10–20 mg of liver tissues were homogenized in 1 mL TRIzol (Thermo Fisher Scientific, Shanghai, China) on ice, incubated for 5 min, then 200 μL chloroform was added. After low-temperature centrifugation, 500 μL isopropanol was used to precipitate RNA. The precipitate was washed with 75% ethanol (DEPC water configuration) and then dissolved in 30 μL DEPC water (Shanghai Generay Biotech Co., Ltd, Shanghai, China) after evaporating all ethanol. The RNA concentration, purity, and integrity were measured by Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Beijing, China). The qualified RNA was reverse transcribed into cDNA and sequenced by BGISEQ-500RS (Beijing Genomics Institute, Beijing, China). The original data were aligned and annotated, referring to GRCh38.p12 Homo sapiens genome. And the FPKM (fragments per kilobase of exon model per million mapped reads) value was used as the standardized numerical output gene expression matrix for data analysis.