Construction of Synthetic Nanobody Library in Mammalian Cells by dsDNA-Based Strategies
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ABSTRACT:
PROVIDER: PRJNA666932 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12762967_1.fastq.gz | Fastqsanger.gz | |||
| SRR12762967_2.fastq.gz | Fastqsanger.gz | |||
| SRR12762968_1.fastq.gz | Fastqsanger.gz | |||
| SRR12762968_2.fastq.gz | Fastqsanger.gz | |||
| SRR12762969_1.fastq.gz | Fastqsanger.gz |
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Project description:A nanobody is an antibody fragment consisting of a single monomeric variable antigen-binding domain. Mammalian cells are ideal platforms for identifying nanobodies targeting hard-to-display transmembrane proteins and nanobodies that function as modulators of cellular phenotypes. However, the introduction of a high-diversity nanobody library into mammalian cells is challenging. We have developed two novel methods for constructing a nanobody library in mammalian cells. Complementarity-determining region (CDR) random sequences were first incorporated into upstream and downstream dsDNAs by PCR. In the first method, named dsDNA-HR, upstream and downstream dsDNAs containing an identical overlapping sequence were co-transfected into cultured mammalian cells for intracellular homologous recombination that resulted in the formation of an intact nanobody library expression cassette. In the second method, named in vitro ligation, we generated full-length nanobody expression dsDNAs via ligation of restriction digested upstream and downstream dsDNAs. The obtained full-length dsDNAs were transfected into mammalian cells for nanobody library expression. Using both methods, we generated over a million unique nanobody sequences, as revealed by high-throughput sequencing. Single-cell sequencing was employed to resolve the diversity of the dsDNA-HR nanobody library. We also identified a small molecule, Nocodazole, which could enhance the efficacy of dsDNA-HR.
2021-09-21 | GSE158906 | GEO
Project description:Nanobody is one special type of single-domain antibody fragment with multiple advantages over traditional antibody. Our previous work established linear-double-stranded DNA (ldsDNA, or PCR amplicon) as novel biological parts for building AND gate genetic circuits in mammalian cells. During this AND-gate circuit formation process, the co-transfected up- and down-stream ldsDNAs could be linked together to form intact gene expression cassette. Here, we employed this ldsDNA-based AND-gate (LBAG) strategy to construct nanobody library in mammalian cells. The sequence complexity of complementary determining regions (CDRs) was introduced into ldsDNA by PCR amplification. After being co-transfected into mammalian cells, the up- and down- stream ldsDNAs undergo AND gate linkage and form full nanobody coding regions, containing CDR1-3. High throughput sequencing identified 22,173 unique oligonucleotide sequences in total generated by this strategy. Thus, we developed a novel method to construct nanobody library, which is a start point for building high content nanobody library in mammalian cells.
2021-07-24 | GSE155159 | GEO
Project description:Construction of Nanobody Library in Mammalian Cells by Linear-double-stranded DNA Based AND Gate Genetic Circuit
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Project description:Low-input Hi-C library construction
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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.
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Project description:Next-generation sequencing has been widely used for the genome-wide profiling of histone modifications, transcription factor binding and gene expression through chromatin immunoprecipitated DNA sequencing (ChIP-seq) and cDNA sequencing (RNA-seq). Here, we describe a versatile library construction method that can be applied to both ChIP-seq and RNA-seq on the widely used Illumina platforms. Standard methods for ChIP-seq library construction require nanograms of starting DNA, substantially limiting its application to rare cell types or limited clinical samples. By minimizing the DNA purification steps that cause major sample loss, our method achieved a high sensitivity in ChIP-seq library preparation. Using this method, we achieved the following: (1) generated high-quality epigenomic and transcription factor-binding maps using ChIP-seq for murine adipocytes; (2) successfully prepared a ChIP-seq library from as little as 25 pg of starting DNA; (3) achieved paired-end sequencing of the ChIP-seq libraries; (4) systematically profiled gene expression dynamics during murine adipogenesis using RNA-seq; and (5) preserved the strand specificity of the transcripts in RNA-seq. Given its sensitivity and versatility in both double-stranded and single-stranded DNA library construction, this method has wide applications in genomic, epigenomic, transcriptomic and interactomic studies. Pre-adipocytes and mature adipocytes were collected. Their chromatin and RNA were subjected to ChIP and mRNA extraction. Sequencing libraries from ChIP DNA or mRNA were generated following either standard protocols or TELP method. The quality and features of TELP libraries were proved and demonstrated in comparison with standard libraries or other published data.
2015-12-15 | E-GEOD-75966 | biostudies-arrayexpress
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2015-03-19 | E-MTAB-3424 | biostudies-arrayexpress
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.
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Project description:Reduction of rRNA during sRNA library construction using blocking oligos
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