The sequencing data were imported as paired-ends into CLC Genomics Workbench 5.5.1 (CLC bio, Aarhus, Denmark) for downstream processing and analysis. Initial trimming of reads was performed using a maximal allowed ambiguity number of 2 within reads, 0.05 limit on quality score, and a removal of the initial 10 5 prime base pairs. Assembly of reads was performed against an annotated Homo sapiens Hg19 reference genome
Sequencing was performed on the Illumina HiSeq2000 platform (Illumina).
The total RNA was isolated using PicoPure RNA isolation kit (Life Technologies) according to the manufacturers protocol, including an additional step of DNase I treatment (Sigma-Aldrich, St. Louis, MO), and stored at -140C until use. The integrity and concentration was determined in the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) using the Agilent RNA 6000 Pico Kit (Agilent Technologies).
An otherwise normal eye suffering from malignant melanoma of the retinal pigment epithelium layer was procured, and the corneo-limbal region was isolated and processed immediately after enucleation at the operation theatre to preserve the integrity of material.
nucleic acid library construction protocol
The total RNA was reverse-transcribed and amplified using Ovation RNA-Seq System V2 kit (NuGEN Technologies, San Carlos, CA), and after quantification of dsDNA by the Qubit kit (Life Technologies), the libraries were constructed for paired-end sequencing with the aid of a TruSeq DNA Sample Preparation Kit (Illumina, San Diego, CA). The libraries were quantified by a real-time PCR and further purified employing Agencourt AMPure XP beads (Beckman Coulter, Brea, CA). Finally, to assure optimal loading for cluster generation, the extracted libraries were quantified using KAPA Library Quant Kits (KAPA Biosystems, Woburn, MA).
Tissue blocks were processed into 10 um serial sections using an HM 505 N (Microm International, Germany) cryostat and mounted on Superfrost Plus glass slides (Thermo Fisher Scientific, Waltham, MA) for phase contrast inspection of niche structures. The selected sections were then transferred onto UV irradiated (3000 uJ/cm2) PEN (polyethylene naphtalate)-membrane metal-framed slides (Life Technologies, Naerum, Denmark), and fixed in 70% ethanol at -30C for 2 min. Staining procedure was completed inside a laminar flow bench using RNase-free pap-jars (Evergreen Scientific, Los Angeles, CA) and reagents pre-cooled to 4C. In short, the slides were washed with water, counter-stained with 0.01% cresyl violet for 10 sec, and washed with 70% ethanol for 30 sec. Finally, the preparations were dehydrated with 100% ethanol for 2 min and xylene for 5 min. The slides were kept inside the pap-jars at room temperature to avoid condensation of water, which could activate endogenous RNases and interfere with LCM. Laser capture microdissection and RNA isolation. The stained tissue sections were sandwiched with Superfrost Plus glass slides to provide a support for dissection and capture in a Veritas 704 microdissection system (Arcturus Bioscience, Mountain View, CA), which was equipped with IR capture and UV cutting lasers. The particular arrangement of the specimen (inverse technique) offered distinctive benefits in minimizing the risk of contamination and enabling large capture area and constant laser settings. The efficiency of capture thus attained 100%, and the hands-on time to finalize the procedure was reduced greatly. The settings for IR laser pulse were power 100 mW, duration 2.7 msec, and hit frequency 1. The UV laser was set on a constant low power. Four discrete compartments were targeted, the basal limbal crypts (BLCs), the superficial limbal crypts (SLCs), the paracentral/central corneal epithelium, and the adjacent limbal stroma. Each of the four specific areas from a single tissue cryosection was comprehensively sampled and collected on individual CapSure Macro LCM Caps (Life Technologies). Single capture cap was used for up to three cryosections and altogether 15 cryosections were processed. To keep the exposure to less than 1 hour, only three specimens were handled at a time, and upon completion, the thermoplastic film overlaid samples were immediately stripped from the caps to be used for RNA extraction.