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ABSTRACT:
PROVIDER: PRJNA750488 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Transposase assisted tagmentation of RNA/DNA hybrid duplexes
| PRJNA600433 | ENA
Project description:Smart-seq3xpress was carefully optimized and >1,000 conditions were evaluated. This data submission is organized in 15 datasets that each contain fastq files, unmapped bam files, read count tables, UMI count tables and a barcode annotation file. The barcode_annotation.txt files contain the exact factors/variables tested. Below a short description of each set of experiments: K562_lowvolume: Evaluation of scaling volumes of Smart-seq3 (indicated volume refers to total volume in PCR), whether overlay was used and if cDNA was bead-cleaned or diluted prior to tagmentation. Cell input was K562 cells. The columns \\"treatment\\", \\"volume\\" and \\"VL\\" indicate the experimental parameters. HEK_lowvolume: Evaluation of scaling volumes of Smart-seq3 (indicated volume refers to total volume in PCR), whether overlay was used and if cDNA was bead-cleaned or diluted prior to tagmentation. Cell input was HEK293FT cells. The columns \\"treatment\\", \\"volume\\" and \\"VL\\" indicate the experimental parameters. overlays: Evaluation of the effect of various overlays when generating HEK293FT libraries in 1 uL total volume. The column \\"condition\\" indicates the applied overlay. tagmentations: Evaluation of input cDNA vs Tn5 amount during tagmentation. Purified cDNA from one 384-well plate was used as input into various conditions of tagmentations. The experiments contain evaluation of cDNA amount with fixed Tn5 amount or varying Tn5 amount while keeping the default volume (2 uL) of the tagmentation reaction or scaling the reaction volume. The column \\"condition\\" contains a string indicating reaction volume, cDNA input and Tn5 ATM enzyme amount. If no volume is indicated, reaction was performed in 2 uL. HomeTn5: Evaluation of tagmentation using in-house produced Tn5 enzyme (Picelli et al., 2015) when tagmenting cDNA generated from HEK293FT cells in 1 uL total volume. The column \\"Tn5concentration\\" indicates the Tn5 reaction concentration at 2 uL reaction volume. cycles_cleanups: Optimization of Smart-seq3xpress (column \\"experiment\\" shows \\"direct_tag\\") in regards to clean-ups after cDNA synthesis (column \\"condition\\": noclean, Exo+FastAP, ExoSAP) and dilution volume (9 or 19 uL); PCR cycle numbers (column \\"pcr_input\\") and ATM Tn5 enzyme amount (column \\"ATM\\"). Cell input was HEK293FT cells. PreAmp_Polymerase: Evaluation of various PCR polymerases during initial cDNA amplification. The polymerases are indicated in column \\"polymerase\\". We also evaluated several TSO concentrations (concentration in RT is given) and fwd/rev PCR primers (concentration given in PCR reaction). Cell input was HEK293FT cells. TDE1: Large optimization of tagmentation conditions using the TDE1 Tn5 enzyme. We varied reactions by changing PCR polymerase (KAPA / SeqAmp), PCR extension time and the number of PCR cycles during cDNA amplification. During tagmentation, we varied the amount of TDE1 enzyme, the amount of DMF in the tagmentation reaction buffer and the presence of Tween-20 in the final post-tagmentation PCR. Cell input was HEK293FT cells. TSOs_RT_v1-7: Large scale evaluation of conditions relating to RT and PCR, with a focus on new template-switching oligo (TSO) designs. In total, >20,000 cells and >500 conditions are contained in these datasets. The barcode annotation file contains precise information on the reaction conditions of Lysis, RT, PCR as well as utilized TSO designs. Data was generated from HEK293FT cells and hPBMC (Lonza).
2022-05-16 | E-MTAB-11467 | biostudies-arrayexpress
Project description:Investigations of 5-hydroxymethylcytosine (5hmC) in biologically and clinically samples and models with low cell numbers have been hampered by the low sensitivity and reproducibility using current 5hmC mapping approaches. Here, we develop a selective 5hmC chemical labeling approach using tagmentation-based library preparation in order to profile nanogram levels of 5hmC isolated from ~1,000 cells (nano-hmC-Seal). Using this technology, we profiled the dynamics of 5hmC across different stages of mouse hematopoietic differentiation. Additionally, applying nano-hmC-Seal to the hematopoietic multipotent progenitor cells in an acute myeloid leukemia (AML) mouse model, we identified leukemia-specific, differentially hydroxymethylated regions that harbor previously reported and as-yet-unidentified functionally relevant factors. The change of 5hmC patterns in AML strongly correlates with the altered gene expression on a global scale. Together, our new approach offers a highly sensitive and robust method to study and detect DNA methylation dynamics from in vivo model and clinical samples. Selective 5hmC chemical labeling approach using tagmentation-based library preparation in order to profile nanogram levels of 5hmC isolated from ~1,000 cells
2016-09-01 | E-GEOD-77967 | biostudies-arrayexpress
Project description:Here we present an enhanced gas phase fractionation (GPF)-assisted DIA-MS acquisition method called PulseDIA, which improves the specificity and sensitivity of Orbitrap-based DIA analysis.
2020-05-24 | PXD019379 | iProX
Project description:Tn5-mediated transposition of double-strand DNA has been widely utilized in various high-throughput sequencing applications. Here, we report that the Tn5 transposase is also capable of direct tagmentation of RNA/DNA hybrids in vitro. As a proof-of-concept application, we utilized this activity to replace the traditional library construction procedure of RNA sequencing, which contains many laborious and time-consuming processes. Results of Transposase assisted RNA/DNA hybrids Co-tagmEntation (termed “TRACE-seq”) are comparable to traditional RNA-seq methods in terms of detected gene number, gene body coverage, gene expression measurement, library complexity, and differential expression analysis; at the meantime, TRACE-seq enables a one-tube library construction protocol and hence is more rapid (within 6h) and convenient. We expect this tagmentation activity on RNA/DNA hybrids to have broad potentials on RNA biology and chromatin research.
2020-07-29 | GSE143422 | GEO
Project description:ngs2012_02_microd-ngs microd-part2-Effect of dilution on RNA seq sensitivity-One sample of total RNA extracted from whole embryo by laser assisted microdissection was diluted from 5ng to 10pg to check sensitivity of the library construction and subsequent sensitivity of transcript identification by RNA seq.
2018-02-13 | GSE98313 | GEO
Project description:Chromatin accessibility mapping is a powerful approach to identify potential regulatory elements. In the popular ATAC-seq method, Tn5 transposase inserts sequencing adapters into accessible DNA (‘tagmentation’). CUT&Tag is a tagmentation-based epigenomic profiling method in which antibody tethering of Tn5 to a chromatin epitope of interest profiles specific chromatin features in small samples and single cells. Here we show that by simply modifying the tagmentation conditions for histone H3K4me2/3 CUT&Tag, antibody-tethered tagmentation of accessible DNA sites is redirected to produce accessible DNA maps that are indistinguishable from the best ATAC-seq maps. Thus, DNA accessibility maps can be produced in parallel with CUT&Tag maps of other epitopes with all steps from nuclei to amplified sequencing-ready libraries performed in single PCR tubes in the laboratory or on a home workbench. As H3K4 methylation is produced by transcription at promoters and enhancers, our method identifies transcription-coupled accessible regulatory sites.
2020-11-13 | GSE158327 | GEO
Project description:Protein-DNA interactions regulate gene expression, and some interactions occur over large distances, such that they are nearby in 3-D space but are separated by many nucleotides in the linear genome. These long-range chromatin loops are essential for gene regulation but remain difficult to interrogate. Methods to capture these chromatin interactions mediated by a specific protein factor include Hi-C sequencing coupled with chromatin immunoprecipitation-sequencing (ChIP-seq), chromatin interaction analysis by paired-end tag sequencing (ChIA-PET), proximity ligation-assisted ChIP-seq (PLAC-seq), and HiChIP. These methods all require high amounts of starting material (0.5M – 100M cells) , which limits their general use. Here, we describe Hi-C Coupled chromatin cleavage and Tagmentation (HiCuT), an enzyme-based tagmentation strategy that provides efficient and high-resolution protein-centric chromatin mapping from as few as 5,000 cells. Thus, HiCuT will permit protein-centric 3-D genome binding assessment in rare cell populations that were previously not testable, including primary cells, human tissue samples, and personalized epigenomics.
2022-03-16 | GSE186011 | GEO
Project description:Single-cell RNA-sequencing (scRNA-seq) is valuable for analyzing cellular heterogeneity. Cell composition accuracy is critical for analyzing cell-cell interaction networks from scRNA-seq data because cell abundancy might affect to the network. We developed terminator-assisted solid-phase cDNA amplification and sequencing (TAS-Seq), a scRNA-seq method that relied on terminator, terminal transferase, and nanowell/beads-based scRNA-seq platform, that could acquire scRNA-seq data with higher correlation with flow-cytometric data, gene-detection sensitivity, and robustness than widely-used methods.
2022-07-01 | GSE200090 | GEO
Project description:Single-cell RNA-sequencing (scRNA-seq) is valuable for analyzing cellular heterogeneity. Cell composition accuracy is critical for analyzing cell-cell interaction networks from scRNA-seq data because cell abundancy might affect to the network. We developed terminator-assisted solid-phase cDNA amplification and sequencing (TAS-Seq), a scRNA-seq method that relied on terminator, terminal transferase, and nanowell/beads-based scRNA-seq platform, that could acquire scRNA-seq data with higher correlation with flow-cytometric data, gene-detection sensitivity, and robustness than widely-used methods.
2022-07-15 | GSE180140 | GEO