9fc4582b-e790-4114-a197-78e21c5c6cea - samples
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ABSTRACT:
PROVIDER: EGAD00001006916 | EGA |
REPOSITORIES: EGA
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Project description:Sequencing was performed to assess the ability of Nanopore direct cDNA and native RNA sequencing to characterise human transcriptomes. Total RNA was extracted from either HAP1 or HEK293 cells, and the polyA+ fraction isolated using oligodT dynabeads. Libraries were prepared using Oxford Nanopore Technologies (ONT) kits according to manufacturers instructions. Samples were then sequenced on ONT R9.4 flow cells to generate fast5 raw reads in the ONT MinKNOW software. Fast5 reads were then base-called using the ONT Albacore software to generate Fastq reads.
2019-03-14 | E-MTAB-7778 | biostudies-arrayexpress
Project description:Myelodysplastic syndrome (MDS) is a clonal myeloid neoplasm characterized by ineffective haematopoiesis and cytopenia with frequent epigenetic modifications. Hypomethylating agents (HMA) such as azacitidine (AZA) and decitabine (DEC) are standard therapeutic options in MDS, but drug resistance is not uncommon.To study RNA modification in particular M6A, P39-AZA-S and P39-AZA-R native RNA libraries were prepared using the direct RNA sequencing kit (Oxford Nanopore) following the manufacturer’s protocol (SQK-RNA002). Differential methylated RNA signal called from raw fast5 files were used as input for basecalling and downstream mapping and methylated transcript detection.
2022-06-01 | GSE165064 | GEO
Project description:In this study, based on Nanopore direct RNA-seq where native RNAs are sequenced directly as near full-length transcripts in the 3' to 5' direction, transcription units of the phytopathogen Dickeya dadantii 3937 were validated and transcriptional termination sites were determined. Briefly, D. dadantii cultures were grown in M63 medium supplemented with 0.2% glucose and 0.2% PGA, until the early exponential phase (A600nm = 0.2, condition 1), or the early stationary phase (A600nm = 1.8, condition 2). RNAs were extracted using a frozen acid-phenol method, as previously described (Hommais et al. 2008), and treated successively with Roche and Biolabs DNases. Two samples were prepared: 50 µg of RNAs from each condition were pulled into one sample (sample 1), whereas the other one contained 100 µg of RNAs from condition 2 (sample 2). Both samples were then supplied to Vertis Biotechnologie AG for Nanopore native RNA-seq: total RNA preparations were first examined by capillary electrophoresis. For sample 1, ribosomal RNA molecules were depleted using an in-house developed protocol (recovery rate = 84%), whereas no ribodepletion was performed for sample 2. 3' ends of RNA were then poly(A)-tailed using poly(A) polymerase, and the Direct RNA sequencing kit (SQK-RNA002) was used to prepare the library for 1D sequencing on the Oxford Nanopore sequencing device. The direct RNA libraries were sequenced on a MinION device (MIN-101B) using standard settings. Basecalling of the fast5 files was performed using Guppy (version 3.6.1) with the following settings: --flowcell FLO-MIN106 --kit SQK-RNA002 --cpu_threads_per_caller 12--compress_fastq --reverse_sequence true --trim_strategy rna. Reads smaller than 50 nt were removed. 466 393 and 556 850 reads were generated for sample 1 and 2, respectively.
2022-01-01 | E-MTAB-10482 | biostudies-arrayexpress
Project description:Transposon insertion site sequencing (TIS) is a powerful method for associating genotype to phenotype. However, all TIS methods described to date use short nucleotide sequence reads which cannot uniquely determine the locations of transposon insertions within repeating genomic sequences where the repeat units are longer than the sequence read length. To overcome this limitation, we have developed a TIS method using Oxford Nanopore sequencing technology that generates and uses long nucleotide sequence reads; we have called this method LoRTIS (Long Read Transposon Insertion-site Sequencing). This experiment data contains sequence files generated using Nanopore and Illumina platforms. Biotin1308.fastq.gz and Biotin2508.fastq.gz are fastq files generated from nanopore technology. Rep1-Tn.fastq.gz and Rep1-Tn.fastq.gz are fastq files generated using Illumina platform. In this study, we have compared the efficiency of two methods in identification of transposon insertion sites.
2022-05-30 | E-MTAB-11351 | biostudies-arrayexpress
Project description:Purpose: RNA-seq analysis to evaluate the impact of MED23 knockdown on the transcriptome of HUVEC cells.Methods: Total RNA was isolated with TRIzol from Med23 knockdown HUVECs (n = 3) or control cells (n = 3). cDNA sequencing libraries were prepared with an NEBNext® Ultra™ RNA Library Prep Kit for Illumina. The RNA-Seq FASTQ raw data were trimmed to remove low-quality reads and adapters using Trimmomatic. The trimmed reads were aligned to the mouse reference genome UCSC GRCh37/hg19 with HISAT2. Gene and transcript quantification was performed using Cutdiff. The results of the mapping of the RNA-seq reads, transcript assembly and abundance estimation were reported as fragments per kilobase of exon per million fragments mapped (FPKM).
2021-10-21 | GSE186112 | GEO
Project description:Transcriptome profiling (RNA-Seq) to evaluate the connection the metabolic enzyme UGDH on gene expression. The RNA libraries were prepared and sequenced at University of Houston Seq-N-Edit Core per protocols. Total libraries were prepared with Ovation® Universal Plus mRNA-Seq (NuGen) using 200 ng input RNA. The size selection for libraries were performed using SPRIselect beads (Beckman Coulter) and purity of the libraries were analyzed using the High Sensitivity DNA chip on Bioanalyzer 2100 (Agilent). The prepared libraries pooled and sequenced using NextSeq 500 (Illumina), generating ~15 million 2×76 bp paired-end reads per samples. The RNA-seq raw fastq data were processed with RNA-Seq Express app within the Illumina BaseSpace app suite (www.basespace.illumina.com), which performed alignment with STAR aligner, assignment of aligned reads to genes, and differential gene expression with DESeq2 Analysis was performed comparing controls (shNT) against individual knockdowns (shU1 or shU2). Then, results from both analyses were compared for the intersection to identify commonly altered genes.
2018-08-21 | GSE118772 | GEO
Project description:Purpose: RNA-seq analysis to evaluate the impact of Msx1-overexpression on the transcriptome of C2C12 cells. Methods: Total RNA was isolated with TRIzol from Msx1-overexpressing C2C12 cells (n = 3) or control cells (n = 3). cDNA sequencing libraries were prepared with an NEBNext® Ultra™ RNA Library Prep Kit for Illumina. The RNA-Seq FASTQ raw data were trimmed to remove low-quality reads and adapters using Trimmomatic. The trimmed reads were aligned to the mouse reference genome UCSC GRCm38/mm10 with HISAT2. Gene and transcript quantification was performed using StringTie. The results of the mapping of the RNA-seq reads, transcript assembly and abundance estimation were reported as fragments per kilobase of exon per million fragments mapped (FPKM).
2020-10-02 | GSE150349 | GEO
Project description:Purpose: The goal of this study is to compare NGS-derived transcriptomes of wild type mouse small interstinal organoids and organoids deficient for the enzyme acetyl-coa-carboxylase (ACC) 1 (Acaca). Methods: mRNA profiles were generated from organoids at 24h and 96h upon in vitro deletion of the Acaca gene and the respective non-deleted wild type controls in triplicates. Total RNA was isolated using RNeasy Micro Kit (Qiagen) and libraries were prepared using NEBNext Single Cell/Low Input RNA Library Prep (New England BioLabs). The libraries were sequenced on Illumina NovaSeq 6000 using NovaSeq 6000 S2 Reagent Kit (100 cycles, paired end run). Each FASTQ file gets a quality report generated by FASTQC tool. Before alignment to reference genome each sequence in the raw FASTQ files were trimmed on base call quality and sequencing adapter contamination using Trim Galore! wrapper tool. Reads shorter than 20 bp were removed from FASTQ file. Trimmed reads were aligned to the reference genome (GRCm38.) using open source short read aligner STAR (https://code.google.com/p/rna-star/) with settings according to log file. Results: the sequencing depth of our libraries is > 3 x107 reads per RNA sample. PCA analysis revealed a close relationship between WT and ACC1-deficient organoids at 24h, whereas samples at 96h were distinct from each other. Lack of ACC1 strongly reduced the expression of genes associated with intestinal epitelial stem cells. Moreover, GSEA revealed downregulation of pathways associated with DNA replication, cell cycle and chromosome segregation in ACC1-deficient organoids. Conclusions: Our study highlights the importance of ACC1-mediated cellular fatty acid synthesis for the maintenance of intestinal epithelial stem cells in mouse intestinal oganoids.
2021-11-10 | GSE188386 | GEO
Project description:Preparation of libraries for circular RNA sequencing with Oxford Nanopore long-read sequencing
2022-08-09 | GSE197872 | GEO
Project description:Purpose:To understand the change in cellular metabolism and function for the overxepression of SCL27A5 in hepatoma cells. Methods:Total RNAs of AdSLC27A5- or AdGFP-infected PLC/PRF/5 cells were extracted using TRIzol (Invitrogen), following the manufacturer’s instructions. RNA-seq and bioinformatic data analysis were performed by Shanghai Novel Bio Ltd. Briefly, strand-specific RNA-seq libraries were prepared using the Total RNA-seq (H/M/R) Library Prep Kit (Vazyme Biotech, Nanjing, China) and were sequenced on a HiSeq X Ten sequencing platform. Raw reads in FASTQ format were subjected to quality control using FastQC. RNA-seq reads were aligned to the reference genome using Bowtie. Uniquely mapped reads were used for further analysis. Gene expression levels are expressed as RPKM (reads per kilobase per million reads) and differences in gene expression were calculated with rSeq. Results:There were 17 genes differentially expressed in AdSLC27A5-infected PLC/PRF/5 cells compare to the GFP control group (fold change >1.5 or < 0.667; FDR < 0.05).
2023-07-01 | GSE117823 | GEO