signature=617db62922ae73f0398aed6e77c8576a,Genome-wide RNA-Sequencing analysis identifies a distinct...

Patient Recruitment

We prospectively recruited glaucoma patients at the Moorfields Eye Hospital (London, UK) from September 2014 to September 2015, and collected conjunctival tissues at the time of glaucoma filtration surgery. All experimental protocols were approved by the London-Dulwich research ethics committee (REC reference 10/H0808/127) and the institutional approval committee at the University College London Institute of Ophthalmology. All the methods were carried out in accordance with the approved guidelines. All patients also gave written informed consent and the study adhered to the tenets of the Declaration of Helsinki. The inclusion criteria were: age (over 18 year-old) and patients planned to have glaucoma tube surgery. The exclusion criteria were previous conjunctival surgery other than glaucoma surgery. Patients were divided into 2 groups: patients who had previous glaucoma surgery and patients with no previous glaucoma surgery.

Clinical Phenotype

We collected detailed clinical data on each patient, including age, ethnicity, gender, best-corrected visual acuity, intraocular pressures, cup-disc ratio, previous glaucoma surgeries, and anti-glaucoma medications. We also assessed each patient using the Moorfields bleb grading system. Central and maximal bleb areas and bleb height were graded on a scale of 1 to 5 (1 = 0%, 2 = 25%, 3 = 50%, 4 = 75%, 5 = 100%). Bleb vascularity was graded on a scale of 1 to 5 (1 = avascular, 2 = normal, 3 = mild hyperaemia, 4 = moderate hyperaemia, 5 = severe hyperaemia).

Fibroblast Cell Lines

We established fibrotic fibroblast (FF) and non-fibrotic fibroblast (NF) primary cell lines from conjunctival tissues collected from patients with previous glaucoma surgery and patients with no previous glaucoma surgery, respectively. The conjunctival tissues were mechanically dispersed and the tissue fragments were placed in tissue culture dishes with Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen), 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine at 37 °C with 5% CO2 as previously described

RNA Sequencing

The RNA was extracted from each fibroblast cell line using the RNeasy mini kit (Qiagen, UK) according to the manufacturer’s instructions. The RNA sequencing was performed at the UCL Genomics facility (London, UK) using TruSeq RNA Library Prep kit v2 (http://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/truseq-rna-v2.html). RNA quality was measured using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA) and each sample was graded using an RNA integrity number equivalent (RINe) between 1 and 10. We used 500 ng of total RNA as input, based on quantification by the Agilent TapeStation RNA assay. Libraries were multiplexed into a single pool and sequenced across all four lanes of an Illumina NextSeq 500. Paired-end sequences of 43 nucleotides were generated after adapter removal.

Differential Expression Analysis

The FASTQ files were assessed for quality control using FASTQC 0.11.2 (http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/). Adapter sequences were removed using Trimgalore (0.43) (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). Paired-end sequence reads were aligned to the human GRCh38.p3 release 82 reference transcriptome and genome using STAR 2.4.2ahttp://www.broadinstitute.github.io/picard) and subsequently removed using Samtoolsp values were adjusted for multiple testing, as part of the DESeq2 analysis, using Benjamini-Hochberg correctionp value 

Gene Ontology, KEGG, Disease association, Pathway commons, and WikiPathways Analyses

We used the list of differentially expressed genes to perform GO (gene ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes), disease association, pathway commons, and WikiPathways analyses using WebGestalt

Protein Network Analysis

The set of genes that were differentially expressed (with an adjusted p value 

Real-Time quantitative PCR

RT-qPCR reactions were performed using a Platinum quantitative PCR master mix (ThermoFisher Scientific, Hemel Hempstead, UK) on a CFX Real-Time PCR detection system (Bio-Rad, Hemel Hempstead, UK). The Taqman gene expression assays were: MYOCD (Hs00538071_m1), IL-6 (Hs00985639_m1), IL-33 (Hs00369211_m1), WISP2 (Hs00180242_m1), PRG4 (Hs00981633_m1), CD34 (Hs02576480_m1), RELB (Hs00232399_m1), IGFBP5 (Hs00181213_m1), MMP-10 (Hs00233987_m1), COL6A6 (Hs01029204_m1), LMO3 (Hs00998696_m1), and GAPDH (Hs02758991_g1) (ThermoFisher Scientific, Hemel Hempstead, UK). All mRNA values were normalised relative to that of GAPDH and triplicate experiments were performed for each gene. Statistical analysis was performed using the Student’s t-test to calculate statistically significant differences and individual p values. The validation of the RNA-Seq data with RT-qPCR was performed using the Spearman’s correlation of the mean values obtained from all samples normalised to either NF or FF samples. Statistically significant differences were expressed as *p