GSM7734299: Viral_S2; Homo sapiens; RNA-Seq
A total of 14 corneal specimens from 14 patients who underwent corneal transplantation at the Eye Center of the University of Freiburg, Germany, were included in this study (Table 1). The indication for surgery was made by an experienced cornea consultant. 7 healthy corneas from 7 patients who underwent enucleation due to choroidal melanoma at our institution served as controls. After surgery, corneal tissue was immediately fixed in formalin and processed for paraffinembedding followed by routine histopathological examination. The clinical diagnosis was confirmed histologically by at least two experienced ophthalmic pathologists (Suppl. Figure 1). There was no evidence of tumor-induced alterations in any of the healthy corneal specimens. Fifteen FFPE sections, each 4 µm thick, were taken from each corneal sample and stored in RNase-free tubes prior to RNA extraction RNA extraction from corneal samples was performed by a commercial provider (GenXPro, Frankfurt am Main, Germany). Briefly, total RNA was isolated from FFPE samples using the Quick-RNA FFPE Kit (Zymo Research, Irvine, CA, USA). Following a DNAse I digestion using the Baseline-ZERO kit (Epicentre, Madison, WI, USA), the RNA concentration was measured with the Qubit RNA HS Assay Kit on a Qubit Fluorometer (Life Technologies, Carlsbad, CA, USA). The RNA quality was determined with the RNA Pico Sensitivity Assay on a LabChip GXII Touch (PerkinElmer, Waltham, MA, USA). RNA sequencing was performed by GenXPro using massive analysis of cDNA ends (MACE), a 3′-RNA sequencing method, as previously described (Wolf et al., 2022c). We recently demonstrated that MACE allows sequencing of FFPE samples with high accuracy (Boneva et al., 2020). Briefly, barcoded libraries comprising unique molecule identifiers were sequenced on the NextSeq 500 (Illumina, San Diego, CA, USA) with 1 × 75 bp. PCR bias was removed using unique molecular identifiers.