Genome-wide detection and quantitation of RNA distribution by ChIRC13a-seq

Eukaryotic genome are transcribed extensively, but a majority of transcripts remain functionally uncharacterized. This is most ascribed to lacking of a potent RNA-centric technology that is capable of accurately quantitating putative genomic binding sites for endogenous RNAs. We describe here a detailed protocol for Chromatin Isolation by RNA-Cas13a Complex sequencing (ChIRC13a-seq), based on recently discovered CRISPR-Cas13a from Leptotrichia wadei (LwaCas13a), for profiling of RNA associated chromatin binding cites. ChIRC13a-seq employs biotinylated, enzymatically-dead Cas13a (dCas13a) that is still capable of binding target RNA and guide RNAs (gRNAs) specific for the RNA target of interest to enrich RNA and its chromatin binding sites. This assay can be performed in standard molecular biology laboratories with 2 d taken for ChIRC13a-seq library preparation.

High-throughput single-cell joint analysis of histone modifications and gene expression by Paired-Tag

We describe here Paired-Tag, a high-throughput multi-omics method for joint profiling of histone modifications and gene expressions in single cells. The assay is based on a combinatorial barcoding indexing strategy that does not require special instruments. It can be performed with nuclei extracted from cultured cells or frozen tissues, in standard molecular biology laboratories.

High-throughput single-cell joint analysis of histone modifications and gene expression by Paired-Tag

We describe here Paired-Tag, a high-throughput multi-omics method for joint profiling of histone modifications and gene expressions in single cells. The assay is based on a combinatorial barcoding indexing strategy that does not require special instruments. It can be performed with nuclei extracted from cultured cells or frozen tissues, in standard molecular biology laboratories.

Biological Nanopores: Engineering on Demand

Nanopore-based sensing is a powerful technique for the detection of diverse organic and inorganic molecules, long-read sequencing of nucleic acids, and single-molecule analyses of enzymatic reactions. Selected from natural sources, protein-based nanopores enable rapid, label-free detection of analytes. Furthermore, these proteins are easy to produce, form pores with defined sizes, and can be easily manipulated with standard molecular biology techniques. The range of possible analytes can be extended by using externally added adapter molecules. Here, we provide an overview of current nanopore applications with a focus on engineering strategies and solutions.

An Efficient Method for Isolation of Plasmid DNA for Transfection of Mammalian Cell Cultures

In this article, we present several protocols that describe the steps from cloning and obtaining a large amount of pure plasmid DNA to generation of lentiviruses based on these constructs. The protocols have been worked out on human cell culture HEK293T but can be adapted for other cell cultures. This protocol was designed to be simple to execute and cheap since it requires only materials and consumables widely available in molecular laboratories, such as salts, alcohols, etc., and no complicated laboratory equipment. These protocols are highly effective and can be performed in any standard molecular biology laboratory.

A Rapid and Accurate MinION-Based Workflow for Tracking Species Biodiversity in the Field

Genetic markers (DNA barcodes) are often used to support and confirm species identification. Barcode sequences can be generated in the field using portable systems based on the Oxford Nanopore Technologies (ONT) MinION sequencer. However, to achieve a broader application, current proof-of-principle workflows for on-site barcoding analysis must be standardized to ensure a reliable and robust performance under suboptimal field conditions without increasing costs. Here, we demonstrate the implementation of a new on-site workflow for DNA extraction, PCR-based barcoding, and the generation of consensus sequences. The portable laboratory features inexpensive instruments that can be carried as hand luggage and uses standard molecular biology protocols and reagents that tolerate adverse environmental conditions. Barcodes are sequenced using MinION technology and analyzed with ONTrack, an original de novo assembly pipeline that requires as few as 1000 reads per sample. ONTrack-derived consensus barcodes have a high accuracy, ranging from 99.8 to 100%, despite the presence of homopolymer runs. The ONTrack pipeline has a user-friendly interface and returns consensus sequences in minutes. The remarkable accuracy and low computational demand of the ONTrack pipeline, together with the inexpensive equipment and simple protocols, make the proposed workflow particularly suitable for tracking species under field conditions.

A rapid and accurate MinION-based workflow for tracking species biodiversity in the field

Genetic markers (DNA barcodes) are often used to support and confirm species identification. Barcode sequences can be generated in the field using portable systems based on the Oxford Nanopore Technologies (ONT) MinION platform. However, to achieve a broader application, current proof-of-principle workflows for on-site barcoding analysis must be standardized to ensure reliable and robust performance under suboptimal field conditions without increasing costs. Here we demonstrate the implementation of a new on-site workflow for DNA extraction, PCR-based barcoding and the generation of consensus sequences. The portable laboratory features inexpensive instruments that can be carried as hand luggage and uses standard molecular biology protocols and reagents that tolerate adverse environmental conditions. Barcodes are sequenced using MinION technology and analyzed with ONTrack, an original de novo assembly pipeline that requires as few as 500 reads per sample. ONTrack-derived consensus barcodes have high accuracy, ranging from 99,8% to 100%, despite the presence of homopolymer runs. The ONTrack pipeline has a user-friendly interface and returns consensus sequences in minutes. The remarkable accuracy and low computational demand of the ONTrack pipeline, together with the inexpensive equipment and simple protocols, make the proposed workflow particularly suitable for tracking species under field conditions.

Swabs to genomes: a comprehensive workflow

The sequencing, assembly, and basic analysis of microbial genomes, once a painstaking and expensive undertaking, has become almost trivial for research labs with access to standard molecular biology and computational tools. However, there are a wide variety of options available for DNA library preparation and sequencing, and inexperience with bioinformatics can pose a significant barrier to entry for many who may be interested in microbial genomics. The objective of the present study was to design, test, troubleshoot, and publish a simple, comprehensive workflow from the collection of an environmental sample (a swab) to a published microbial genome; empowering even a lab or classroom with limited resources and bioinformatics experience to perform it.

Swabs to genomes: a comprehensive workflow

The sequencing, assembly, and basic analysis of microbial genomes, once a painstaking and expensive undertaking, has become almost trivial for research labs with access to standard molecular biology and computational tools. However, there are a wide variety of options available for DNA library preparation and sequencing, and inexperience with bioinformatics can pose a significant barrier to entry for many who may be interested in microbial genomics. The objective of the present study was to design, test, troubleshoot, and publish a simple, comprehensive workflow from the collection of an environmental sample (a swab) to a published microbial genome; empowering even a lab or classroom with limited resources and bioinformatics experience to perform it.

Swabs to Genomes: A Comprehensive Workflow

The sequencing, assembly, and basic analysis of microbial genomes, once a painstaking and expensive undertaking, has become almost trivial for research labs with access to standard molecular biology and computational tools. However, there are a wide variety of options available for DNA library preparation and sequencing, and inexperience with bioinformatics can pose a significant barrier to entry for many who may be interested in microbial genomics. The objective of the present study was to design, test, troubleshoot, and publish a simple, comprehensive workflow from the collection of an environmental sample (a swab) to a published microbial genome; empowering even a lab or classroom with limited resources and bioinformatics experience to perform it.