biostudies-arrayexpressPiotr KarabowiczHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16859Study investigates the biological role of small RNAs present in aqueous humor in patients with primary open-angle glaucoma, pseudoexfoliative glaucoma, and cataract, with the aim of identifying molecular biomarkers and improving understanding of disease mechanisms. Aqueous humor samples were collected during ophthalmic surgery, followed by RNA extraction from the cell-free supernatant using a commercial kit designed for low-input RNA. Small RNA sequencing libraries were prepared using adapter ligation with Unique Molecular Identifiers, reverse transcription, PCR amplification, and magnetic bead purification, and library quality was assessed using a microfluidics-based system. The pooled libraries were sequenced using paired-end sequencing on an Illumina NextSeq platform, and the resulting FASTQ files were used for downstream bioinformatic and machine learning analyses integrating molecular, clinical, and imaging data.biostudies-arrayexpressLibrary Construction - The total yield of purified RNA from each patient was fully employed to generate sequencing libraries using the high-sensitivity, low-input TrueQuant Small RNA-Seq Library Preparation Kit (GenXPro GmbH, Frankfurt, Germany), adhering meticulously to the manufacturer’s protocol. Briefly, small RNA molecules were ligated to 3’ and 5’ adapters containing TrueQuant Unique Molecular Identifiers (UMIs) (TrueQuant Adapters; GenXPro GmbH, Frankfurt, Germany), followed by reverse transcription and polymerase chain reaction (PCR) amplification with sample-specific indexed primers. Amplification was conducted with a reduced cycle number to minimize bias, succeeded by purification via magnetic bead-based separation. The resultant libraries underwent quality evaluation using a DNA analysis cartridge (Agilent High Sensitivity DNA Kit; Agilent Technologies, Santa Clara, CA, USA) on a microfluidics-based system (Agilent Bioanalyzer 2100; Agilent Technologies).Nucleic Acid Extraction - The samples of aqueous humor were thawed slowly on ice and centrifuged at 3000g for 15 minutes at 4°C to remove cells and cell debris, with the supernatant collected for the extraction of RNA. The supernatant were completed up to 200 µl volume with a 10 mM Tris-HCL pH 8.0 buffer, and RNA was extracted using a commercial kit for RNA purification (miRNeasy Serum/Plasma Advanced Kit; Qiagen, Hilden, Germany), according to the manufacturer’s recommendations.Sequencing - The verified libraries were pooled and analyzed through paired-end sequencing, generating 2 × 65 base pair reads, on a next-generation sequencing platform (NextSeq 2000; Illumina, Inc., San Diego, CA, USA).Sample Collection - Aqueous humor samples were procured from patients slated for elective glaucoma or cataract surgeries. Intraoperative collection was performed via ab-externo limbal paracentesis using a 27-gauge needle attached to a tuberculin syringe. Rigorous precautions were instituted to prevent vascular interference and to ensure the integrity of the samples against contamination. A volume ranging from 50 to 200 microliters of aqueous humor was aspirated into a cryovial prior to commencing the primary corneal incision, after which the samples were promptly stored in dry ice within the operating theater. Specimens exhibiting evidence of hemorrhage or failing to meet the minimum volume threshold of 50 microliters were excluded from subsequent analysis. Successfully collected and preserved aqueous humor samples—immediately snap-frozen post-extraction—were transported in batches under stringently controlled conditions (maintained frozen on dry ice) to the molecular analysis laboratory.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Raw sequencing reads were subjected to adapter trimming and quality filtration using Cutadapt (version 4.6). Deduplication was executed based on Unique Molecular Identifiers (UMIs) employing a dedicated software suite (TrueQuant Software; GenXPro, Frankfurt, Germany). Sequence integrity was assessed with FastQC (version 0.11.9). Processed reads were subsequently mapped to the hg38 (Homo sapiens) reference genome using Bowtie2 (version 2.4.4), aligned against an array of RNA databases, including miRNA (miRBase), tRNA (GtRNAdb), piRNA (piRNAdb), ncRNA (ENSEMBL), and cDNA (ENSEMBL) This alignment proceeded iteratively, with unmapped reads from one database progressively aligned to the next. Quantification of reads corresponding to specific transcripts was performed using HTSeq (version 2.0.2).UnknownTranscriptomicsGenomicsProteomicsNextSeq 2000RNA-seq of coding RNAHomo sapiensPiotr KarabowiczfalseSmall RNA-seq Aqueous humor of human glaucoma (open-angle glaucoma and pseudoexfoliative glaucoma) patients against cataractStudy investigates the biological role of small RNAs present in aqueous humor in patients with primary open-angle glaucoma, pseudoexfoliative glaucoma, and cataract, with the aim of identifying molecular biomarkers and improving understanding of disease mechanisms. Aqueous humor samples were collected during ophthalmic surgery, followed by RNA extraction from the cell-free supernatant using a commercial kit designed for low-input RNA. Small RNA sequencing libraries were prepared using adapter ligation with Unique Molecular Identifiers, reverse transcription, PCR amplification, and magnetic bead purification, and library quality was assessed using a microfluidics-based system. The pooled libraries were sequenced using paired-end sequencing on an Illumina NextSeq platform, and the resulting FASTQ files were used for downstream bioinformatic and machine learning analyses integrating molecular, clinical, and imaging data.2026-04-30T00:00:00Z2026-04-30T01:01:55.368Z2026-04-07T11:09:05.725ZE-MTAB-16859ERP191807EFO_0002944EFO_0004170EFO_0005518EFO_0003816EFO_0003738EFO_0004184