#Transcribe dna free

This decreases the proportion of reads that map to the nuclear genome, reducing the amount of information that can be used to identify regulatory regions of open chromatin. However, a drawback of this protocol is that the hyperactive Tn5 transposase also targets sources of extranuclear genetic material, including the genomes of mitochondria and chloroplasts. ATAC-seq is a fast protocol with simple library amplification steps and requires very small amounts of starting material, making it a vast improvement over alternative methods. This method uses a hyperactive Tn5 transposase to integrate preloaded sequencing adapters into regions of open chromatin ( Fig. ДЄn improved method for identifying accessible regions of chromatin and transcription factor binding is the Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq). Alternatively, regulatory regions can be inferred by Chromatin Immunoprecipitation sequencing (ChIP-seq, see Chapter 5) where antibodies are used to pull down transcription factors or histone marks associated with active transcription. Characterizing such regulatory regions throughout the genome has therefore relied on methods that combine enzymatic digestion of nuclear DNA and high-throughput sequencing, such as microccocal nuclease sequencing (MNase-seq, see Chapter 10) and DNase I Hypersensitivity sequencing (DNase-seq). Binding of transcription factors to DNA generally results in the depletion of nucleosomes, rendering these regions hypersensitive to nucleases. Transcription is associated with regions of relatively open chromatin, in which cis-regulatory elements such as enhancers and promoters can recruit transcription factors and RNA polymerase II to transcribe DNA. Plants are sessile organisms that must precisely regulate their transcription in response to their environment, as well as for proper development, growth, and homeostasis.

The procedures described here are optimized for Arabidopsis thaliana but can also be applied to other plant species. Sequencing-ready ATAC-seq libraries can be prepared from plant tissue in as little as one day. We provide detailed instructions for transposase treatment of nuclei isolated using either approach, as well as subsequent preparation of ATAC-seq libraries. Here we present two different approaches to purify plant nuclei for ATAC-seq: the INTACT method (Isolation of Nuclei TAgged in Specific Cell Types) to isolate nuclei from individual cell types of the plant, and tissue lysis followed by sucrose sedimentation to isolate sufficiently pure total nuclei.

#Transcribe dna free

The application of ATAC-seq to plant tissue has been challenging due to the difficulty of isolating nuclei sufficiently free of interfering organellar DNA. This technique utilizes a hyperactive Tn5 transposase to cause DNA cleavage and simultaneous insertion of sequencing adapters into open chromatin regions of the input nuclei. In this chapter we describe an approach to map accessible chromatin features in plants using the Assay for Transposase Accessible Chromatin, combined with high throughput sequencing (ATAC-seq), which was originally developed for cultured animal cells. Identifying and characterizing highly accessible chromatin regions assists in determining the location of genomic regulatory elements and understanding transcriptional regulation.