Transcriptome Analysis of Primary Orbital Fibroblasts

The total RNA of primary orbital fibroblasts was extracted using an RNA Purification Kit (Yishan Biotechnology, Shanghai, China). RNA extracts were analyzed using an RNA Nano 6000 Assay Kit (Agilent Technologies, CA, USA) in combination with the Bioanalyzer 2100 system. RNA sample preparations were carried out with a total amount of 1 μg RNA per sample. NEBNext^® UltraTM RNA Library Prep Kit (Illumina, CA, USA) was used for library preparation and index codes were added to each sample as attribute specifier sequence. After sample clustering, the library preparations were sequenced on an Illumina Novaseq platform. Quality control raw reads were processed using in-house Perl scripts. The reference genome index was built using Hisat2 (v2.0.5) and the paired-end reads were aligned against the reference genome using Hisat2 (v2.0.5) as well. The mapped reads of each sample were assembled by StringTie (v1.3.3b) (29 (link)). Differential expression testing was performed using the DESeq2 R package (1.20.0). Genes with a P-value < 0.05 as identified by DESeq2 were considered differentially expressed.