Ena-DATASET-LUMC-SASC-16-11-2017-14:49:46:788-791 - samples
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PROVIDER: EGAD00001003806 | EGA |
REPOSITORIES: EGA
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Project description:The Arabidopsis seedlings treated with 2M t-zeatin (CTK) for 30 minutes and then total RNA was extracted from Arabidopsis seedlings using TRIzol reagent (Invitrogen) and treated with RNase-free DNase I. The poly(A) mRNA was isolated using oligo (dT) beads. First-strand cDNA was subsequently generated using random hexamer-primed reverse transcription, followed by synthesis of the second-strand cDNA using RNaseH and DNA polymerase I. Then, single-end and paired-end RNA-seq libraries were prepared following Illumina's protocols and sequenced on the Illumina GA II platform.
2016-06-17 | E-MTAB-4603 | biostudies-arrayexpress
Project description:mRNA transcriptome sequencing of tumor bearing lungs from KrasLSL-G12D/+Lkb1fl/fl, KrasLSL-G12D/+Lkb1fl/flIl1f9-/-(KL9) and KrasLSL-G12D/+Lkb1fl/flIl1f5-/-(KL5) or KrasLSL-G12D/+Tp53fl/fl, KrasLSL-G12D/+Tp53fl/flIl1f9-/-(KP9) and KrasLSL-G12D/+Tp53fl/flIl1f5-/-(KP5) after 10 weeks Ad-cre injection Tumor-burdened lungs from KL/KL5/KL9 mice and KP/KP5/KP9 mice were perfused through alveolar lavage and cardiac lavage with PBS, dry it quickly, and then homogenized in 2 ml of TRIzol (Invitrogen). Total RNAs were prepared and the quality of RNAs was determined by agarose gel electrophoresis and spectrophotometer analysis. Poly(A) mRNA was subsequently purified from 10μg total RNA using NEBNext Oligo d(T)25 Magnetic Beads Isolation Module. First-strand complementary DNA was synthesized with NEBNext RNA First-Strand Synthesis Module. NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module was used for the synthesis of the complementary strand of first-strand cDNA. The resulting double-stranded DNA was purified and Vazyme TruePrep DNA Library Prep kit V2 was used to prepare libraries followed by sequencing on an Illumina Hiseq X Ten platform with 150-bp paired-end reads strategy (Novogene). Quality control of mRNA-seq data was performed by using Fatsqc (v0.11.9) and low-quality bases were trimmed by Trim_galore (0.6.4_dev). All RNA-seq data were mapped to the mouse genome (Mus_musculus_Ensemble_94) by Hisat2 (v.2.0.5) and allowed a maximum of two mismatches per read. Gene expression level was calculated by FeatureCounts (v.2.0.0) with default parameters and normalized by FPKM (Fragments Per Kilobase of exon model per Million mapped fragments).
2021-01-28 | GSE165640 | GEO
Project description:Il1f5+/+, Il1f5-/-, Il1f9+/+ and Il1f9-/- mice were administered 2.5% DSS dissolved in sterile water for 5 days, followed by regular water for 2 days. The colons were flushed with PBS to clear feces and slit open longitudinally. The distal colon tissues (0.5 cm in length, about 0.5 cm away from the anus) were washed in PBS and homogenized in 2 ml TRIzol (Invitrogen). Colorectal tumors from Il1f5+/+, Il1f5-/-, Il1f9+/+ and Il1f9-/- mice were AOM/DSS colorectal tumor model. The colorectal tumors were flushed with PBS to clear feces and homogenized in 2 ml of TRIzol (Invitrogen). Total RNAs were prepared and the quality of RNAs was determined by agarose gel electrophoresis and spectrophotometer analysis. Poly(A) mRNA was subsequently purified from 10μg total RNA using NEBNext Oligo d(T)25 Magnetic Beads Isolation Module. First-strand complementary DNA was synthesized with NEBNext RNA First-Strand Synthesis Module. NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module was used for the synthesis of the complementary strand of first-strand cDNA. The resulting double-stranded DNA was purified and Vazyme TruePrep DNA Library Prep kit V2 was used to prepare libraries followed by sequencing on an Illumina Hiseq X Ten platform with 150-bp paired-end reads strategy (Novogene). Quality control of mRNA-seq data was performed by using Fatsqc (v0.11.9) and low-quality bases were trimmed by Trim_galore (0.6.4_dev). All RNA-seq data were mapped to the mouse genome (Mus_musculus_Ensemble_94) by Hisat2 (v.2.0.5) and allowed a maximum of two mismatches per read. Gene expression level was calculated by FeatureCounts (v.2.0.0) with default parameters and normalized by FPKM (Fragments Per Kilobase of exon model per Million mapped fragments).
2021-02-06 | GSE166277 | GEO
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: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:We performed the cleavage under targets and tagmentation (Cut & tag) assay followed by sequencing enriched DNA fragments to reveal the direct downstream targets of Pbx1. Firstly, we overexpressed Pbx1b with Pbx1b-IRES-GFP retrovirus in murine peripheral B cells to ensure the yields of DNA fragments. CUT & tag libraries were generated following instructions of the manufacturer’s protocol (Vazyme; cat TD901-01) and the Pbx1 antibody (CST; cat 4342) was used for signal enrichment.
2023-03-26 | E-MTAB-12769 | biostudies-arrayexpress
Project description:T cells from OT-I mice were stimulated with 4 different peptide ligands for 6 hours and sorted into two 96-well plates. Cells on each plate were barcoded using a mutually exclusive 8-by-12 set of indexes, such that indexes present on plate 1 were completely absent from plate 2 and vice versa. Libraries from each plate were pooled in equimolar quantities and sequenced on an Illumina HiSeq 4000. Libraries were demultiplexed allowing for all pairs of indexes, including the expected combinations (pairs of barcodes used within each plate) and unexpected combinations (pairs containing one barcode from each plate). This was repeated after sequencing the same pool of libraries on the HiSeq 2500.
2018-06-21 | E-MTAB-6843 | biostudies-arrayexpress
Project description:The influenza polymerase cleaves host RNAs ~10–13 nucleotides downstream of their 5′ ends and uses this capped fragment to prime viral mRNA synthesis. To better understand this process of cap snatching, we used high-throughput sequencing to determine the 5′ ends of A/WSN/33 (H1N1) influenza mRNAs. The sequences provided clear evidence for nascent-chain realignment during transcription initiation and revealed a strong influence of the viral template on the frequency of realignment. After accounting for the extra nucleotides inserted through realignment, analysis of the capped fragments indicated that the different viral mRNAs were each prepended with a common set of sequences and that the polymerase often cleaved host RNAs after a purine and often primed transcription on a single base pair to either the terminal or penultimate residue of the viral template. We also developed a bioinformatic approach to identify the targeted host transcripts despite limited information content within snatched fragments and found that small nuclear RNAs and small nucleolar RNAs contributed the most abundant capped leaders. These results provide insight into the mechanism of viral transcription initiation and reveal the diversity of the cap-snatched repertoire, showing that noncoding transcripts as well as mRNAs are used to make influenza mRNAs. Sixteen datasets were generated, corresponding to template-switching 5’-RACE libraries of 1) each viral RNA, 2) a time-course of NS1 viral RNA, or 3) CIP-TAP libraries of NS1 and PB2. Libraries contain random barcodes of length 5 (NS1_TS, PB2_TS, NS1_5R, PB2_5R) or 9 (other datasets) at the start of each read, and at least three guanosine residues for the template-switching datasets. See methods of accompanying paper for further details. Samples were sequenced on an Illumina HiSeq 2000.
2015-04-23 | E-GEOD-65101 | biostudies-arrayexpress
Project description:This track depicts high throughput sequencing of long RNAs (>200 nt) from RNA samples from tissues or subcellular compartments from ENCODE cell lines. The overall goal of the ENCODE project is to identify and characterize all functional elements in the sequence of the human genome. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Cells were grown according to the approved ENCODE cell culture protocols. Sample preparation and sequencing: K562 and GM12878 total cell, total RNA: Standard Illumina Pair-end kit with the sole exception that a "tagged" random hexamer was used to prime the 1st strand synthesis: 5'-ACTGTAGGN6-3'. The addition of this tag is what permits us to make strand assignments for the reads. The sequence of the tag is reported in the 5' end of the read. Asymmetric PCR can place the tag on either the 1st or 2nd read depending on which strand it used as a template. Strand assignments are made by looking for the tag at the 5' end of either read 1 or read 2. Read 1 is physically linked to read 2. Therefore, if a tag is present on one end strand assignments are made for both ends. We noted during analysis that the tags are generally 5' truncated. We only "strand" reads that contain ACTGTAGG, CTGTAGG, TGTAGG, GTAGG. Between 63-68% of reads could be stranded in these libraries. It is possible to cull additional stranded reads that contain non-templated TAGG, AGG, GG, or G sequences at their 5' end. The peak in insert size distribution is between 200-250 nucleotides. K562 cytosol, polyA+ RNA: Oligo-dT selected poly-A+ RNA was RiboMinus-treated according to the manufacturer's protocol (Invitrogen). The RNA was treated with tobacco alkaline pyrophosphatase to eliminate any 5' cap structures and hydrolyzed to ~200 bases via alkaline hydrolysis. The 3' end was repaired using calf intestinal alkaline phosphatase, and poly-A polymerase was used to catalyze the addition of Cs to the 3' end. The 5' end was phosphorylated using T4 PNK, and an RNA linker was ligated onto the 5' end. Reverse transcription was carried out using a poly-G oligo with a defined 5' extension. The inserts were then amplified using oligos targeting the 5' linker and poly-G extension. This cloning protocol generated stranded reads that were read from the 5' ends of the inserts. The library was sequenced on a Solexa platform for a total of 36 cycles; however, the reads underwent post-processing, resulting in trimming of their 3' ends. Consequently, the mapped read lengths are variable. Analysis: K562 and GM12878 total cell, total RNA: Tags were removed from the 5' ends of the reads in accordance to their lengths and strand assignments made. Subsequently, the reads were trimmed from their 3' ends to a final length of 50 nucleotides and were mapped using NexAlign, a program developed by Timo Lassman, RIKEN. We allowed up to 2 mismatches across the entire length and only report reads that mapped to a single/unique locus in the assembled hg18 genome. K562 cytosol, polyA+ RNA: Reads were mapped to the human (hg18, March 2006) assembly using Nexalign, with only uniquely mapping (one loci), exactly matching (no mis-matches) aligned reads reported in the processed files, as follows: 1) Collect the read sequences from Illumina non-filtered output files. 2) Filter out all reads that contain undefined nucleotides ('N'). 3) Perform iterative alignment/C-tail chopping algorithm (below). On each alignment step, the reads are aligned to the genome with 100% identity. All reads that align to a single locus are withdrawn from the alignment pool and only the reads that could not be aligned continue to the next step. a) Align to the hg18 genome using Nexalign 1.3.3 (© Timo Lassmann) without chopping off any nucleotides. b) Chop off any C-blocks (until the first non-C) at the ends of the reads. c) Align to the genome -> remove and save those that align. d) Chop off any non-Cs until the next C. e) Chop off C-block until the next non-C. f) Align to the genome -> remove and save those that align. g) Repeat steps d, e, and f until the reads align to the genome, or chopping results in the reduction of the reads' lengths to below 16 (default), or there are no non-Cs left.
2011-10-12 | E-GEOD-32931 | biostudies-arrayexpress
Project description:The tracks show enrichment of RNA sequence tags mapped to the mouse genome generated by high throughput sequencing (RNA-Seq). Double stranded cDNA was synthesized from enriched RNA that was obtained after depletion of ribosomal RNA. Pieces of cDNA, 300-350 nucleotides in length, were PCR amplified, adapter ligated, and sequenced on an Illumina HiSeq sequencer. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Cells were grown according to the approved ENCODE cell culture protocols (http://hgwdev.cse.ucsc.edu/ENCODE/protocols/cell). Total RNA was extracted using RNeasy Mini Kit (74104, Aiagen), following the manufacturer's protocol. Ribosomal RNA was removed from total RNA using the Ribo-ZeroTM Gold Kits (MRZG126, Epicentre). Double-stranded cDNA synthesis was performed on the rRNA depleted RNA using random primers and the SuperScript double-stranded cDNA synthesis kit (11917-010, Life Tech). After first strand cDNA synthesis, NucAway Spin Column (Ambion cat. 100070-30) was used to remove dNTPs. In the second strand cDNA synthesis reaction, dTTP in the dNTP mix was substituted with dUTP. After end repair and addition of 'A' base to 3' end, illumina paired-end adapter was ligated to Double-stranded cDNA library. After gel size selection of adapter ligated cDNA (300-350), Uracil-N-Glycosylase (UNG: Applied Biosystems) was used to digest the second strand cDNA (Parkhomchuk et al. , 2009). PCR amplified adapter ligated cDNA was sequenced using Illumina HiSeq. Sequence reads of 2x101 nt long with 0-2 mismatches were mapped to the mouse genome (version mm9) using the BWA aligner, version 0.5.7. The signal height corresponds to the number of overlapping fragments at each nucleotide position in the genome.
2012-07-24 | E-GEOD-39619 | biostudies-arrayexpress