Ena-DATASET-SickKids-17-12-2020-21:20:54:099-50 - samples
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PROVIDER: EGAD00001006813 | EGA |
REPOSITORIES: EGA
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Project description:Purpose: The goal of this study is to complare the transcitpional profiles of freshly isolated, splenic, murin GCBC and other activated and naive non-germinal center B cell populations. Methods: Cells were freshly isolated, bead purified and FACS Ari sorted . Sorted cells (1.5-3x106 cells per sample) were washed twice in cold PBS and RNA was prepared using shredder-columns (Qiagen; QIAshredder 79656) and the RNeasy Plus Mini kit (Qiagen) following the manufacturer’s instructions. 3 independent RNA libraries per cell population were prepared using Illumina TruSeq Stranded mRNA sample preparation kit. The first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T oligo attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity is achieved by using dUTP in the Second Strand Marking Mix, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. The products are then purified and enriched with PCR to create the final cDNA library. The cDNA libraries are validated using KAPA Biosystems primer premix kit with Illumina-compatible DNA primers and Qubit 2.0 fluorometer. Quality is examined using Agilent Tapestation 2200.The cDNA libraries were pooled at a final concentration 1.8pM. Samples were sequenced using Illumina NextSeq 500 with 75bp paired-end reads and aligned to the mm10 genome using the STAR aligner. The number of uniquely aligned reads ranged from 31 to 77 million. Using an optimized data analysis workflow, Gene-level counts were determined using featureCounts and raw counts were analyzed for differential expression using the “voom” method in the “limma” R package. Results: After determining genes that were differentially expressed between splenic GCBC and activated B cells, we performed gene set enrichment analysis (GSEA). Differentially expressed genes were compared to several previously defined hypoxic gene signatures.
2020-01-01 | GSE128710 | GEO
Project description:Inhibition of ZNF768 function was achieved by conditional over expression expression of the C-terminal zinc finger of ZNF768 for 12h. For preparation of total RNA cells were resuspended in TRIzol reagent (Life Technologies) at 0.9Mio/ml and snap-frozen. After thawing RNA was extracted from 0.4ml of TriZol lysate using the direct-zol RNA Miniprep (Zymo Research, Irvine CA, USA) as described in the manufacturer’s protocol. RNA was assessed for purity by UV-vis spectrometry (Nanodrop) and for integrity by Bioanalyzer (Agilent Bioanalyzer 2100, Agilent, Santa Clara USA)). RNA was of high purity (abs. 260/280 >1.9, abs 269/239>2.1) and integrity (Bioanalyzer RIN>9 ) and thus used for further processing. For production of RNA-seq libraries total RNA was DNAse treated (dsDNAse, Fermentas) and 100 ng of this RNA was processed with a strand-specific protocol (RNA-seq complete kit, NuGEN, San Carlos, USA). In brief the RNA was reverse transcribed to cDNA with a reduced set of hexamer primers, avoiding excessive representation of rRNA in the cDNA. Second strand cDNA synthesis was done in presence of dUTP. After ultrasonic fragmentation of the cDNA and end repair, Illumina-compatible adapter were ligated. Adapters contained uracil in one strand, allowing complete digestion of the second-strand derived DNA. After strand selection the libraries were amplified, assessed for correct insert size on the Agilent Bioanalyser and diluted to 10nM. Barcoded libraries were mixed in equimolar amounts and sequenced on an Illumina HiSeq1500 in single-read mode with a read length of 100 b.
2018-10-29 | GSE111881 | GEO
Project description:Purpose: To comprehensively identify traits controlled by the PhcA quorum sensing system, and to better understand how this regulator affects pathogen behavior in its natural host environment Method: 21 day-old tomato plants were inoculated with approximately 2000 cells of either wild-type Rs strain GMI1000 or ΔphcA through a cut petiole. After 72 h in a 28°C growth chamber (12h day/night cycle), 0.1 g of stem tissue spanning the petiole was harvested and transferred immediately to a tube containing 900 µl of pre-chilled transcription stop solution (5% [vol/vol] water-saturated phenol in ethanol) and ground using a Powerlyzer. RNA was isolated from these ground samples using hot phenol method. Total RNA was treated with RiboZero kits to reduce bacterial and plant rRNA, followed by tomato mRNA reduction using polyA selection and used to make libraries with the Illumina TruSeq strand-specific mRNA sample preparation system. After RNA fragmentation, strand-specific libraries were constructed by first-strand cDNA synthesis using random primers, sample cleanup and second-strand synthesis using DNA Polymerase I and RNase H. A single 'A' base was added to the cDNA fragments followed by ligation of the adapters. The final cDNA library was further purified and enriched with PCR, and quality checked using the Bioanalyzer 2100. The libraries were sequenced using the Illumina HiSeq2500, yielding ~20 million 1x50bp reads per sample Conclusion: Defining the PhcA in planta regulon identified traits that make R. solanacearum successful at high cell density during bacterial wilt disease, but also identified traits that help it survive at low cell densities
2017-09-08 | GSE98074 | GEO
Project description:Purpose: Compare the transcriptional profile of staurosporine-treated cells in Neurospora crassa wild type cells grown with different availabilities of calcium. Methods: Conidial suspensions were obtained and 1 x 106 cells/ml incubated in minimal medium (with the indicated concentrations of calcium) for 6 hours (26ºC, 140 rpm, constant light) followed by the addition of staurosporine (or DMSO) and growth for 1 more hour. For the experiments in 0 Ca2+ or 20 mM CaCl2 media, the concentration of KH2PO4 in the 50x Vogel’s stock solution was limited to 10 mM to avoid precipitation with supplemental calcium. Cells were harvested using 0.45 μm filters and immediately frozen in liquid nitrogen. Total RNA was isolated by the Trizol-Phenol-Chloroform method. After digestion of 25 μg RNA with TURBO DNAse (Life Technologies), mRNA was purified using Dynabeads oligo(dT) magnetic beads (Life Technologies). The mRNA was chemically fragmented using the Ambion RNA fragmentation kit (Life Technologies). First and second strand cDNA synthesis was achieved using appropriate kits (Life Technologies). The illumina TruSeq kit was employed to generate the cDNA libraries with indexing adapters essentially following the manufacturer’s protocol. After purification of the libraries with AMPure XP beads (Roche), the quality of the libraries was analyzed in a Agilent 2100 Bioanalyzer. The cDNA libraries were sequenced in a illumina HiSeq2000 and single reads of 50 bp were obtained. Sequencing data was handled essentially with Tophat, Cufflinks and Cuffdiff. Expression levels are presented as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Results: Differences in the concentration of extracellularly available Ca2+ result in distinct transcriptional programs. Conclusions: Our transcriptomic analysis provides a reference dataset for future investigations on the role of Ca2+ in fungal biology.
2014-12-05 | GSE53013 | GEO
Project description:RNA from A673 cells with shRNA-mediated knockdown of GFP (4 libraries), EWS-FLI1 (4 libraries), or lnc277 (7 libraries) was isolated with TRIzol (Invitrogen). Each sample was DNase treated and further purified on an RNeasy Mini column (Qiagen) before quality analysis on an Agilent 2100 Bioanalyzer. For each sample, 100-150ng of RNA was synthesized into cDNA, sheared on a Covaris ultrasonicator, and amplified using the NuGen Encore Complete kit (NuGen) to produce strand-specific and rRNA-depleted libraries. Samples were multiplexed (4/lane) for 2x100bp paired-end sequencing on an Illumina HiSeq 2000
2014-11-01 | GSE60949 | GEO
Project description:We report the use to RNA-sequencing to understand the role of postnatal Arx in the regulation of PV interneuron function. Total RNA extracted from control and Arx CKO male mice cells were submitted to the Next-Generation Sequencing Core of the University of Pennsylvania for cDNA amplification, library preparation, and sequencing using the Ovation RNA-seq v2 and Rapid DR Multiplexing kits (NuGEN). Whole mRNAseq libraries were prepared from 9 mice (5 control and 4 Arx CKO mice) using the SMARTer Ultra Low RNA Kit for Illumina Sequencing (Clontech, Cat#634936) according to manufacturer’s instructions. Standard methods to assess sample quality (Agilent Bioanalyzer, Kappa Biosystems Library Quantification kit, Qubit® Fluorometer) and measure cDNA concentration were used. A total of 1ng of amplified cDNA for each sample was used as input to build the sequencing libraries. Whole mRNAseq libraries from control and Arx CKO mice were quantified using Agilent Bioanalyzer DNA 7500 chips. Libraries were sequenced on the Illumina HiSeq 4000 2 × 100 (Illumina, San Diego, CA, USA) to generate 150bp paired end reads, yielding approximately 20-40 million reads per sample (~80 million reads per lane).
2020-09-09 | GSE157689 | GEO
Project description:Purpose: We used GAS as a model bacterial pathogen to investigate the complex relationship between genome, transcriptome, and virulence in a large population of type emm28 strains recovered from humans over 26 years in geographically diverse locations. Methods: We chose a subset of isolates from the sequenced population of 2,101 emm28 strains that would approximately span the genetic variation present in the entire population. 442 emm28 strains grown as singleton cultures and harvested at one time point (early-stationary phase), and libraries were prepared using RNAtag-seq. Quality of the total RNA, rRNA-depleted RNA, and cDNA libraries was evaluated with RNA Nano, RNA Pico, and DNA high-sensitivity kits (Agilent Technologies), respectively, using an Agilent 2100 Bioanalyzer. For each sample, the cDNA library concentration was measured fluorometrically with Qubit™ dsDNA HS assay kits (Invitrogen). The cDNA libraries were diluted, pooled, and sequenced with an Illumina NextSeq 500 instrument.
2018-12-31 | GSE113055 | GEO
Project description:Purpose: Evaluate the transcriptional profile of Neurospora crassa cells treated with a group of novel antifungal compounds Methods: Conidial suspensions were obtained and 1 x 106 cells/ml incubated in minimal medium for 6 hours (26ºC, 140 rpm, constant light) followed by the addition of the indicated compound (or DMSO) and growth for 1 more hour. Cells were harvested using 0.45 μm filters and immediately frozen in liquid nitrogen. Total RNA was isolated by the Trizol-Phenol-Chloroform method. After digestion of 25 μg RNA with TURBO DNAse (Life Technologies), mRNA was purified using Dynabeads oligo(dT) magnetic beads (Life Technologies). The mRNA was chemically fragmented using the Ambion RNA fragmentation kit (Life Technologies). First and second strand cDNA synthesis was achieved using appropriate kits (Life Technologies). The illumina TruSeq kit was employed to generate the cDNA libraries with indexing adapters essentially following the manufacturer’s protocol. After purification of the libraries with AMPure XP beads (Roche), the quality of the libraries was analysed in a Agilent 2100 Bioanalyzer. The cDNA libraries were sequenced in a illumina HiSeq2000 and single reads of 50 bp were obtained. Sequencing data was handled essentially with Tophat, Cufflinks and Cuffdiff. Expression levels are presented as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Results: XP13 promoted a particularly active transcriptional response. Functional enrichment analysis of the genes with altered expression revealed novel pathways that are affected by these compounds. Conclusions: These compounds are active in Neurospora crassa and we anticipate that this and future studies will ascertain their potential as antifungal agents.
2015-11-25 | GSE53040 | GEO
Project description:The goal of this study is to investigate the differential transcripted genes affected by CRISPR induced endoglin knockout in PASMC cells. Total RNA was purified from NTC or ENG-/- PASMC cells using RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA quality and concentration were assessed with Agilent Tapestation 200 (Agilent Technologies) and Qubit 2.0 (ThermoFisher Scientific). ~250-500 ng RNA were used for library construction. The NGS libraries were constructed using the KAPA Stranded mRNA-Seq Kits (KapaBioSystems). mRNA was captured using magnetic oligo-dT beads and 1st strand cDNA was synthesized using random priming. In order to preserve strand-specificity, 2nd strand synthesis, which converts the cDNA:RNA hybrid to double-stranded cDNA (dscDNA), was marked by dUTP incorporation. cDNA framents were A-tailed by adding dAMP to the 3'-ends of the dscDNA library fragments. dsDNA Illumina TruSeq "forkedâ adapters 3'-dTMP overhangs were then ligated to A-tailed library insert fragments. Each of the six libraries were ligated with a unique Truseq 6bp barcode. Library fragments were amplified using the KAPA HiFi HotStart polymerase. The strand marked with dUTP was not amplified, allowing strand-specific sequencing. Fragment length and library quality was assessed on a 2100 Bioanalyzer using the High Sensitivity DNA Kit (Agilent Technologies). Libraries were diluted to 10nM and pooled at equimolar ratios. The pool was then diluted to 2nM and denatured in NaOH following Illumina recommendations. 10pM of denatured library pool was loaded in one HiSeq lane and flowcell was clustered on the Illumina C-bot. 5% PhiX control was spiked-in. The flowcell was sequenced on a HiSeq 2500 V4 chemistry with 50bp Single read protocol. Data was demultiplexed and Fastq files were generated using BcptoFastq 1.8.4 script provided by Illumina.
2017-05-16 | E-GEOD-78271 | biostudies-arrayexpress
Project description:Purpose: Compare the transcriptional profile of staurosporine-treated cells in Neurospora crassa wild type cells grown with different availabilities of calcium. Methods: Conidial suspensions were obtained and 1 x 106 cells/ml incubated in minimal medium (with the indicated concentrations of calcium) for 6 hours (26M-BM-:C, 140 rpm, constant light) followed by the addition of staurosporine (or DMSO) and growth for 1 more hour. For the experiments in 0 Ca2+ or 20 mM CaCl2 media, the concentration of KH2PO4 in the 50x VogelM-bM-^@M-^Ys stock solution was limited to 10 mM to avoid precipitation with supplemental calcium. Cells were harvested using 0.45 M-NM-<m filters and immediately frozen in liquid nitrogen. Total RNA was isolated by the Trizol-Phenol-Chloroform method. After digestion of 25 M-NM-<g RNA with TURBO DNAse (Life Technologies), mRNA was purified using Dynabeads oligo(dT) magnetic beads (Life Technologies). The mRNA was chemically fragmented using the Ambion RNA fragmentation kit (Life Technologies). First and second strand cDNA synthesis was achieved using appropriate kits (Life Technologies). The illumina TruSeq kit was employed to generate the cDNA libraries with indexing adapters essentially following the manufacturerM-bM-^@M-^Ys protocol. After purification of the libraries with AMPure XP beads (Roche), the quality of the libraries was analyzed in a Agilent 2100 Bioanalyzer. The cDNA libraries were sequenced in a illumina HiSeq2000 and single reads of 50 bp were obtained. Sequencing data was handled essentially with Tophat, Cufflinks and Cuffdiff. Expression levels are presented as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Results: Differences in the concentration of extracellularly available Ca2+ result in distinct transcriptional programs. Conclusions: Our transcriptomic analysis provides a reference dataset for future investigations on the role of Ca2+ in fungal biology. Transcriptional profile of staurosporine-treated Neurospora crassa wild type cells growing with different concentrations of calcium was compared using illumina HiSeq2000 and single reads of 50 bp.
2014-12-05 | E-GEOD-53013 | biostudies-arrayexpress