Ena-DATASET-NCCS-07-08-2019-08:18:22:214-290 - samples
Ontology highlight
ABSTRACT:
PROVIDER: EGAD00001005230 | EGA |
REPOSITORIES: EGA
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Project description:Whole-transcriptome sequencing (WTS) of 6 tumor-normal and 6 tumor-only samples from patients diagnosed with angiosarcoma. Total RNA from snap frozen EITL tumor samples was extracted using TRIzol (Invitrogen) and purified with RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. The integrity of RNA was determined by electrophoresis using 2100 Bioanalyzer (Agilent Technologies). 500 ng of total RNA was reverse transcribed with iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Quantification was performed using SsoFast EvaGreen Supermix and CFX96 Real-Time PCR System (both Bio-Rad). Sequencing libraries were prepared using the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero (Illumina) and WTS was performed on HiSeq 2500 and HiSeq 3000 (Illumina) with 2x101 bp and 2x151 bp read length, respectively.
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Project description:WGS and WTS data of a single patient diagnosed with HSTCL.
Whole-genome sequencing (WGS) was performed for the tumor-normal sample. Genomic DNA from tumor tissue was extracted with QIAamp DNA Mini Kit. The DNA for the matching normal was obtained from blood or buccal swabs and purified by Blood and Cell Culture DNA Mini kit or E.Z.N.A. Tissue DNA Kit (Omega Bio-tek) according to manufacturer’s instructions. The quantity and quality were assessed by Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen) and agarose gel electrophoresis. All sequencing libraries were prepared using TruSeq Nano DNA Library Prep Kit (Illumina). Paired-end sequencing was performed on Illumina HiSeq 4000 2x151 bp read length.
Whole-transcriptome sequencing (WTS): Total RNA from snap frozen EITL tumor samples was extracted using TRIzol (Invitrogen) and purified with RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. The integrity of RNA was determined by electrophoresis using 2100 Bioanalyzer (Agilent Technologies). 500 ng of total RNA was reverse transcribed with iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Quantification was performed using SsoFast EvaGreen Supermix and CFX96 Real-Time PCR System (both Bio-Rad). Sequencing libraries were prepared using the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero (Illumina) and WTS was performed on Illumina HiSeq 2500 with 2x101 bp read length.
| EGAD00001005229 | EGA
Project description:WGS from 4 patients, WTS from only 3 patients (insufficient tissue from 4th patient for WTS).
Whole-genome sequencing (WGS) was performed for 60 pairs of tumor-normal samples from patients diagnosed with NKTL. Genomic DNA from tumor tissue was extracted with QIAamp DNA Mini Kit. The DNA for the matching normal was obtained from blood or buccal swabs and purified by Blood and Cell Culture DNA Mini kit or E.Z.N.A. Tissue DNA Kit (Omega Bio-tek) according to manufacturer’s instructions. The quantity and quality were assessed by Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen) and agarose gel electrophoresis. All sequencing libraries were prepared using TruSeq Nano DNA Library Prep Kit (Illumina). Paired-end sequencing was performed on Illumina HiSeq 2000.
Whole-transcriptome sequencing (WTS): Total RNA from snap frozen EITL tumor samples was extracted using TRIzol (Invitrogen) and purified with RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. The integrity of RNA was determined by electrophoresis using 2100 Bioanalyzer (Agilent Technologies). 500 ng of total RNA was reverse transcribed with iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Quantification was performed using SsoFast EvaGreen Supermix and CFX96 Real-Time PCR System (both Bio-Rad). Sequencing libraries were prepared using the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero (Illumina) and WTS was performed on Illumina HiSeq 2500 with 2x101 bp read length.
Description of prefix used in filenames:
T: Tumor samples
N: Normal samples (Blood)
P: PDX samples
| EGAD00001005341 | EGA
Project description:To extract RNA from shoots and roots, 3 weeks old control and treated plants were harvested at 2pm in the greenhouse. Total RNA was extracted using the AccuPrep® Universal RNA extraction kit (Bioneer, Daejeon, Korea) and treated with RNase-free DNase to remove DNA fragments (Qiagen, Hilden, Germany). One microgram of total RNA was used to synthesize cDNA with AccuPower® RT PreMix (Bioneer, Daejeon, Korea). Quantitative real-time RT-PCR was conducted using a T100TM Thermocycler system (Bio-Rad, Hercules, CA, USA). Primer information is given in Table S7. Reactions (10 µL final volume) were prepared using 5 µL of LaboPass™ SYBR Green Q Master Kit (Cosmogenetech, Dajeon, Korea). 0.5 pmol of a primer pair, and 0.5 µL of cDNA template. 4 biological samples and two technical replicates were used for the quantifications. Ubiquitin was used as the reference. gene expression analysis was performed with the 2^−ΔΔCt method using Bio-Rad CFX Maestro software v.4.0 (Bio-Rad). Baseline and threshold levels were set according to the manufacturer’s instructions.
2024-02-04 | GSE248090 | GEO
Project description:Total RNA was extracted using TRI® Reagent (Sigma). cDNA was synthesized by RevertAid™ First Strand cDNA Synthesis Kit with Oligo dT primers (K1622, Fermentas) following manufacturer’s recommendations. PCR reactions were carried out on a DNAEngine® Thermal Cycler (PTC-0200G, Bio-Rad) in 25 μl reaction volume containing 1 μl cDNA, 200 nM primer pairs and components of TaKaRa Taq™ kit (R001A, Takara). All samples were analyzed in triplicate RT-qPCR.mRNAs were extracted using biotinylated poly(dT) oligo, followed by further removing of contaminated rRNA using RiboMinus Transcriptome Isolation Kit (K1550-02, Invitrogen). Then mRNAs were fragmented into 100-200nt length and subjected to immunoprecipitation with m6A specific antibody.The libraries were sequenced using HiSeq2000 (Illumina) in single-read mode, creating reads with a length of 101 bp. Sequencing chemistry v2 (Illumina) was used and samples were multiplexed in two samples per lane.
2015-03-09 | GSE53244 | GEO
Project description:The goal of this study is to identify novel target genes of DVL3 in two breast cancer cell lines Methods:Total RNA was isolated using the Aurum™ Total RNA Mini Kit (Bio Rad) and library preparation and sequencing were performed at Center for Biotechnology & Genomics of Texas Tech University. RNA quality was determined using RNA Screen Tape (Agilent). Ribosomal RNA depletion was achieved using NEB Next rRNA Depletion Kit (Human/Mouse/Rat) (NEB # E6310X). RNA fragmentation, double stranded cDNA and adaptor ligation was generated using NEBNext Ultra II Directional RNA Library Prep according to the manufacturer’s protocol (NEB # E7760L). PCR enriched libraries were quantified by Qubit and equimolar indexed libraries (different samples had different indexes for multiplexing) were pooled. Pooled libraries were quantitatively checked using the Agilent Tapestation 2200 and quantified using Qubit. The libraries were then diluted to 200 pM and spiked with 2% phiX libraries (Illumina control). The transcriptome sequencing was performed on the barcoded stranded RNA-Seq libraries using Illumina NovaSeq 6000 SP flow cell, paired-end reads (2 × 50 bp).
2021-04-16 | GSE165773 | GEO
Project description:Corneal stromal cells from six donors were used and expanded in standard conditions (FBS as a supplement) and xenofree conditions (HPL as a supplement). Bone marrow MSCs from one other donor were used as a comparative control. From the purified RNA, 10ng was used per sample to synthesize cDNA using the iScript Advanced cDNA synthesis kit (Bio-Rad Laboratories, Temse, Belgium) following the protocol supplied by the manufacturer. qRT-PCR assays were prepared in triplicate in 96-well PrimePCR plates for human MSCs (Bio-Rad Laboratories, Temse, Belgium), in which the primers come lyophilized per well. A total of 88 genes was targeted, which account for stemness, differentiation (osteo-, chondro-, adipo-, teno- and myogenesis), MSC-specific, and MSC-related genes. The thermal cycler used to perform the PCR was the CFX96 Real-Time System – C1000 Thermal Cycler (Bio-Rad Laboratories, Temse, Belgium). SsoAdvanced universal SYBR Green supermix was used to quantify double-stranded DNA. Dissociation of double-stranded DNA was assessed in melting curve analysis to determine the specificity. Three reference housekeeping genes were included in the assay plates (TBP, GAPDH and HPRT1) to normalize expression using the ΔΔCq method. The abundance of the transcripts in CS-MSC relative to the control BM-MSC was determined by calculating the relative normalized expression (RNE) or fold change. All analyses were performed using the Bio-Rad CFX Manager software. When gene expression exceeds a more than threefold change compared to BM-MSC it is deemed to be up- or downregulated.
2018-06-08 | GSE115453 | GEO
Project description:RNA was extracted from cells using Aurum Total RNA kit from Bio-Rad Laboratories, Inc., Hercules, CA following the manufacturer’s recommendations. Gene expression profiling was performed using the BeadChip HumanHT-12 v4 Expression kit from Illumina®, which contains 47,231 gene-probes (Illumina® Inc., San Diego, CA). The raw signal intensities were imported and analyzed using the GenomeStudio® data software. After background subtraction and normalization, the signal intensity values were exported to the Partek® genomics expression analysis suite using “Partek's Report Plug-in” option in the GenomeStudio® software. Differentially expressed genes in the dox- versus vehicle-treated samples were identified using the “gene expression” workflow in the Partek® software.
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Project description:Total RNA, greater than 100ng, from cultured cells was isolated in TRIzol L and purified using Qiagen RNeasy Mini Kit per manufacturer’s protocols. Agilent Technologies 2100 Bioanalyzer was used to assess the RNA quality. RNA libraries were prepared and multiplexed using Illumina TruSeq RNA Library Preparation Kit v2 (non-stranded and poly-A selection) and 10 nM of cDNA was used as the input for high-throughput sequencing via Illumina’s HiSeq 2500 platform, producing 50 bp paired end reads.
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