mDNA-seq libraries were prepared using the QIAseq FX DNA library kit (Qiagen, Hilden, Germany) with an index for each sample, followed by short-read sequencing using the NexSeq 500 platform (2 × 150-mer paired-end) (Illumina, San Diego, CA, USA). Adapters and low-quality sequences were trimmed using Sickle version 1.33 (https://github.com/najoshi/sickle ) with the following parameters: average quality threshold “-q 20” and minimum length threshold “-l 40” Subsequently, mDNA-seq analysis was performed using clean reads for homology searches without de novo assembly.
Nexseq500 platform
Manufactured by Illumina
Sourced in United States, United Kingdom, Austria
The NexSeq500 platform is a high-throughput sequencing system designed for a wide range of genomic applications. It utilizes sequencing-by-synthesis technology to generate DNA sequence data. The system is capable of producing large volumes of sequence data in a single run.
Automatically generated - may contain errors
Lab products found in correlation
Qiaseq fx dna library kit, by Qiagen (1 mentions)
Qubit hs kit, by Thermo Fisher Scientific (1 mentions)
Truseq rna sample preparation kit, by Illumina (1 mentions)
Direct zol rna miniprep kit, by Zymo Research (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (1 mentions)
Bioanalyzer rna 6000 nano kit, by Agilent Technologies (1 mentions)
Mirneasy mini kit protocol, by Qiagen (1 mentions)
Rnalater, by Merck Group (1 mentions)
Quantseq fwd kit, by Lexogen (1 mentions)
Qiacube, by Qiagen (1 mentions)
Kapa stranded mrna seq kit, by Roche (1 mentions)
5 protocols using nexseq500 platform
1
Mitochondrial DNA Sequencing Protocol
2
RNA Extraction and Sequencing Protocol
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RNAs for transcriptional profile by sequencing were extracted using the Direct-zol RNA miniprep kit (ZYMO Research, Cambridge) from two independent biological replicates. The extracted RNAs were quantified using Qubit HS kit (Invitrogen, UK) and quality checked using a Bioanalyzer 2100 (Agilent, UK). Total RNA libraries were prepared using the TruSeq RNA Sample Preparation Kit (Illumina, UK) and sequenced in single-end 150 base mode an Illumina NexSeq500 platform at the University of Warwick.
3
Robust RNA Extraction from Rabbit Lungs
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After collection, lungs were immediately placed in RNA Later (Sigma Aldrich, USA) and stored at − 20 °C. Samples were homogenized in QIAzol® Lysis Reagent. Total RNA was extracted with the miRNeasy Mini Kit protocol (QIAGEN, Germany), using an automated method (QIAcube: QIAGEN, Germany) adapted to include DNase I treatment. RNA concentration was measured using Qubit 4 fluorometer (Thermo Fisher Scientific, USA). RNA integrity was assessed by checking the 2:1 ratio of 18S and 28S ribosomal RNA bands and RNA Integrity Number (RIN) by agarose gel electrophoresis and Bioanalyzer RNA 6000 Nano Kit (Agilent, USA), respectively. Lung-derived RNA was suitable for RNA-sequencing (RIN > 8). Libraries for high-throughput RNA sequencing were prepared using the QuantSeq FWD kit (Lexogen, Austria) and sequenced in three different runs with the Illumina NexSeq500 platform, which generated at least 20 million reads for each sample. 96% of the reads were mapped to the rabbit genome.
4
RNA-Seq of Fruit Development and Ethephon
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Two sets of fruit samples were collected for RNA-sequencing: (1) fruit at four developmental/ripening stages: Immature green (IMG), Green, Pink and Ripe (2) control and ethephon treated fruit at 1 and 2 days after treatment. IMG fruit was based on size ranging from > 7-<13 mm in diameter. Green, Pink, and Ripe fruit had two biological replicates, while all remaining samples had three biological replicates. Fruit from control and ethephon treatments were comprised of a random sample with fruit at varying developmental time-points. Each sample was frozen in liquid N2 and then stored at -80 °C until further processing. Six to eight fruit for each sample were ground into fine powder and total RNA was isolated from the tissue using the cetyltrimethyl ammonium bromide (CTAB)-based method described in [77 (link)]. High quality RNA (RIN > 8.0) from each sample was used for RNA-Seq library construction with KAPA Stranded mRNA-Seq Kit (KAPA Biosystems, USA) for the Illumina platform, following the instructions provided in the manufacturer’s manual. Libraries were sequenced at the Georgia Genomics and Bioinformatics Core at UGA using an Illumina NexSeq500 platform with 75 bp paired-end sequencing.
5
Genetic Characterization of MODY Patients
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Fifty-two index cases with suspected MODY were selected. A clinical exome was made using the Gen Exome Panel v1.0 kit on the NexSeq500 platform (Illumina). Data Genomics software (Imegen) was used for the bioinformatics analysis. The clinical characterization of the gene variants was carried out according to the ACMG recommendations. The databases used for the variant analysis were ClinVar and HGMD. The Selene software was used to obtain the medical records. The specific variants were verified by Sanger methodology and the large rearrangements by MLPA (Multiplex Ligation Probe Amplification). The SPSS v27 program was used to compare the phenotypic characteristics between the group 1 (patients carrying a pathogenic variant) and the group 2 (non-carriers patients). Quantitative variables (age of debut, HbA1c, BMI, serum levels of insulin and C-peptide) were compared using the Mann-Whitney U test, and qualitative variables (patients with pancreatic antibodies, insulin treatment and family history) with Fisher's exact test. A value of p <0.05 was considered statistically significant.
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