Ontology highlight
ABSTRACT:
PROVIDER: PRJNA906703 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Comparison of one-step and two-step PCR for 16S Illumina MiSeq
| PRJEB48796 | ENA
Project description:Comparison of one-step and two-step PCR for 16S Illumina MiSeq
| PRJEB43472 | ENA
Project description:Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) and its ultra-high resolution cousin ChIP-exo are methods that identify where proteins bind along any genome in vivo. ChIP-exo achieves near-base pair resolution by creating exonuclease stop sites just 5’ to where formaldehyde-induced protein-DNA cross-links occur. Whereas construction of ChIP genomic libraries is straightforward and widely adopted for ChIP-seq, ChIP-exo is technically more involved which has resulted in limited adoption. Here we describe multiple ChIP-exo protocols, each with use-specific advantages and limitations. The new versions are greatly simplified through removal of multiple enzymatic steps. This is achieved in part through the use of Tn5 tagmentation and/or single-stranded DNA ligation. The result is greater library yields, lower processing time, and lower cost. A similar streamlined approach was developed for ChIP-seq, called ChIP-seq 1-step, where library construction is achieved in one-step.
2018-08-17 | GSE110681 | GEO
Project description:Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) and its ultra-high resolution cousin ChIP-exo are methods that identify where proteins bind along any genome in vivo. ChIP-exo achieves near-base pair resolution by creating exonuclease stop sites just 5’ to where formaldehyde-induced protein-DNA cross-links occur. Whereas construction of ChIP genomic libraries is straightforward and widely adopted for ChIP-seq, ChIP-exo is technically more involved which has resulted in limited adoption. Here we describe multiple ChIP-exo protocols, each with use-specific advantages and limitations. The new versions are greatly simplified through removal of multiple enzymatic steps. This is achieved in part through the use of Tn5 tagmentation and/or single-stranded DNA ligation. The result is greater library yields, lower processing time, and lower cost. A similar streamlined approach was developed for ChIP-seq, called ChIP-seq 1-step, where library construction is achieved in one-step.
2018-08-17 | GSE114606 | GEO
Project description:Analysis of Cas9/sgRNA mutagenic activity at a variety of loci in zebrafish. Each loci has a control, where no Cas9/sgRNA were injected. This is amplicon sequencing with Illumina, after PCR amplification. Data was processed with ampliCan R package version 1.1.1.
2017-12-25 | E-MTAB-6310 | biostudies-arrayexpress
Project description:Analysis of Cas9/sgRNA mutagenic activity at a variety of loci in zebrafish. Each loci has a control, where no Cas9/sgRNA were injected. This is amplicon sequencing with Illumina, after PCR amplification. Data was processed with ampliCan R package version 1.1.1.
2017-12-15 | E-MTAB-6355 | biostudies-arrayexpress
Project description:High-throughput sequencing (HTS) of antibody amplicon libraries has become a powerful method in the emerging field of systems immunology. However, numerous sources of bias in HTS workflows may affect antibody repertoire data. A crucial step in antibody amplicon library preparation is the addition of adapter sequences, which are platform-specific short nucleotide sequences. In addition to conventional ligation-based adapter addition, it is also possible to use PCR-based methods such as Direct Addition (one-step adapter addition, DA) and Primer Extension (two-step adapter addition, PE), which have led to the lack of a consensus method and uncertainty regarding the impact of adapter addition on repertoire HTS data. Therefore, we compared all three methods by performing HTS with the Illumina MiSeq platform using total RNA originating from mouse antibody-secreting cells. We used technical replicate-based validation and clonal overlap and rank statistics to demonstrate that the two PCR-based methods produced HTS repertoires equivalent to ligation. Specifically: Nine BALB/c mice (8-10 weeks old, Charles River, specific-pathogen free) were immunized with 50 ug alum-precipitated chicken gamma globulin (CGG) conjugated to 4-hydroxy-3-nitrophenylacetyl (NP, NP-CGG, BioCat). Mice were sacrificed 14 days post-immunization (dpi) and their spleens and bone marrow (from femurs and tibia) were harvested. Antibody-secreting CD138-positive cells were enriched from spleen and bone marrow 14 dpi as previously described (Reddy, 2010, Nat Biotech).
2014-02-20 | E-MTAB-2226 | biostudies-arrayexpress
Project description:Deep mutational scanning (DMS) makes it possible to perform massively parallel quantification of the relationship between genetic variants and phenotypes of interest. However, the difficulties in introducing large variant libraries into mammalian cells greatly hinder DMS under physiological states. Here we developed two novel strategies for DMS library construction in mammalian cells, namely ‘piggyBac-in-vitro ligation’ and ‘piggyBac-in-vitro ligation-PCR’. For the first strategy, we took the ‘in-vitro ligation’ approach to prepare high-diversity linear dsDNAs, and integrate them into the mammalian genome with a piggyBac transposon system. For the second strategy, we further added a PCR step using the in-vitro ligation dsDNAs as templates, for the construction of high-content genome-integrated libraries via large-scale transfection. Both strategies could successfully establish genome-integrated EGFP-chromophore randomized libraries in HEK293T cells and enrich the green fluorescence-chromophore amino acid sequences. And we further identified a novel transcriptional activator peptide with the ‘piggyBac-in-vitro ligation-PCR’ strategy. Our novel strategies greatly facilitate the construction of large variant DMS library in mammalian cells, and may have great application potential in the future.
2023-10-05 | GSE226719 | GEO
Project description:One-step mutants
| PRJEB78485 | ENA
Project description:Background: Next-generation sequencing (NGS) is fundamental to the current biological and biomedical research. Construction of sequencing library is a key step of NGS. Therefore, various library construction methods have been explored. However, the current methods are still limited by some shortcomings. Results: This study developed a new NGS library construction method, Single strand Adaptor Library Preparation (SALP), by using a novel single strand adaptor (SSA). SSA is a double-stranded oligonucleotide with a 3ʹ overhang of 3 random nucleotides, which can be efficiently ligated to the 3′ end of single strand DNA by T4 DNA ligase. SALP can be started with any denatured DNA fragments such as those sheared by Tn5 tagmentation, enzyme digestion and sonication. When started with Tn5-tagmented chromatin, SALP can overcome a key limitation of ATAC-seq and become a high-throughput NGS library construction method, SALP-seq, which can be used to comparatively characterize the chromatin openness state of multiple cells unbiasly. In this way, this study successfully characterized the comparative chromatin openness states of four different cell lines, including GM12878, HepG2, HeLa and 293T, with SALP-seq. Similarly, this study also successfully characterized the chromatin openness states of HepG2 cells with SALP-seq by using 105 to 500 cells. Conclusions: This study developed a new NGS library construction method, SALP, by using a novel kind of single strand adaptor (SSA), which should has wide applications in the future due to its unique performance.
2018-01-31 | GSE104162 | GEO