title: GSM4408810: CNV replicate 2; Homo sapiens; RNA-Seq
library_construction_protocol: CNV membranes for RNA-Seq were obtained from 4 patients by the currently obsolete technique of subretinal CNV extraction. Following a 20-gauge vitrectomy, we performed retinotomy temporal to the macula and surgically extracted the CNV membrane using a flexed forceps. The diagnosis was made after a thorough funduscopic examination of both eyes. Only patients exhibiting a CNV associated with AMD changes, such as drusen and RPE alterations, were included in the study. CNV membranes from patients with other diseases, such as myopia or a history of central serous chorioretinopathy, were not included in this study. Control RPE/choroid samples for RNA-Seq were obtained from the macula region of four enucleated eyes suffering from ciliary body melanoma. Fifteen 4-µm-thick FFPE sections from each CNV membrane were collected and stored in tubes prior to RNA extraction. FFPE eyes of patients with ciliary body melanoma were melted and halved and the macular region was carefully examined under the microscope. The retina of the macula was removed and the RPE/choroid carefully scraped and stored in tubes until RNA extraction was performed. RNA isolation from FFPE specimens was carried out as previously described. Briefly, total RNA was extracted from FFPE samples using the Quick-RNA FFPE Kit (Zymo Research). Following DNAse I digestion using the Baseline-ZERO Kit (Epicentre), the RNA concentration was quantified using the Qubit RNA HS Assay Kit on a Qubit Fluorometer (Life Technologies). RNA quality was determined via the RNA Pico Sensitivity Assay on a LabChip GXII Touch (PerkinElmer). The fragment size of all RNA samples ranged between 120 and 150 bp. The preparation of massive analysis of cDNA ends (MACE) libraries was carried out by GenXPro GmbH using 1 µg of total RNA as previously described. Briefly, eight barcoded libraries (four CNV membranes and four RPE-choroidal control tissues) were sequenced simultaneously on the NextSeq 500 (Illumina) with 1 × 75 bp.
attributes.tag: GEO Accession
attributes.value: GSM4408810