dna denaturation solutions  (Thermo Fisher)

Journal: Nature protocols

Article Title: Quantitative Assessment of RNA-Protein Interactions with High Throughput Sequencing - RNA Affinity Profiling (HiTS-RAP)

doi: 10.1038/nprot.2015.074

Figure Lengend Snippet: Overview of High Throughput Sequencing-RNA Affinity Profiling (HiTS-RAP). (A) First, recombinant proteins and DNA template need to be generated. A DNA template suitable for Illumina sequencing and presentation of RNA transcripts on the flowcell is required (see Fig. 2). Recombinant Tus protein and fluorescently labeled protein of interest (either as an mOrange fusion or a comparable dye-labeled form) are required for transcription halting and detection of bound protein, respectively. As an optional step, testing of the recombinant proteins and the DNA template for proper function, and detectable interaction between the protein of interest and the RNA transcript of the template DNA is recommended before performing HiTS-RAP. The multi-step process of HiTS-RAP (see Fig. 3) is performed on an Illumina GAIIx instrument yielding sequence and protein binding information for about 200 million RNAs on a single flowcell. Finally, HiTS-RAP generated sequencing and binding data are analyzed to determine the protein binding affinity (Kd) of each RNA presented on the flowcell (see Fig.4). A timeline for HiTS-RAP is provided, where generous estimates of time that each step would take for a new user are indicated on the left side. The whole process can be completed in 2–3 weeks. (B) An Illumina Genome Analyzer IIx (GAIIx) equipped with Paired-End Module (PEM) and controlled by a Dell T7500 workstation, is used for HiTS-RAP. The main function of each component is indicated below the schematics with green text. At its core, the GAIIx is both an automated microfluidic device, which can precisely control amounts, incubation times, and temperature of reagents delivered to the flowcell (red parallelogram), and a sensitive Total Internal Reflection Fluorescence (TIRF) microscope, which can quantitatively detect multiple fluorescent entities on the surface of the flowcell. The GAIIx is easily programmable. These features make the GAIIx an attractive instrument to repurpose for applications other than sequencing, as has been done in HiTS-FLIP, RNA-MaP, and HiTS-RAP. Delivery of reagents to the flowcell inside the instrument is indicated by a cyan arrow. HiTS-RAP is fully automated; an unmodified GAIIx programmed with a single user-edited recipe performs every step of HiTS-RAP sequentially, and requires minimal user input once the instrument is set up (see Fig. 3)

Article Snippet: , , Low or high DNA template concentration , Make sure the DNA template was properly quantified and diluted, as both higher and lower amounts leading to under- or over-clustering on the flowcell, respectively, can yield lower than ideal sequences in GAIIx. A 20 pM denatured DNA solution, as recommended by Illumina, can be used for cluster generation on flowcell using cBot..

Techniques: Next-Generation Sequencing, Recombinant, Generated, Illumina Sequencing, Labeling, Sequencing, Protein Binding, Binding Assay, Control, Incubation, Fluorescence, Microscopy

Journal: Nature protocols

Article Title: Quantitative Assessment of RNA-Protein Interactions with High Throughput Sequencing - RNA Affinity Profiling (HiTS-RAP)

doi: 10.1038/nprot.2015.074

Figure Lengend Snippet: DNA template and RNA transcript of HiTS-RAP. (A) Schematics of the DNA template used for HiTS-RAP and the resulting halted RNA transcript. DNA template encoding the RNA of interest (green) is flanked by the Illumina flowcell adaptor 1 (gray) and T7 RNA polymerase promoter (orange) upstream, and by the Illumina sequencing primer annealing site (purple), Tus-binding Ter site (red), and Illumina flowcell adaptor 2 downstream. Illumina flowcell adaptors 1 and 2 are required for cluster generation on Illumina GA flowcell. T7 RNA polymerase promoter is required for transcription of the RNA of interest. The Illumina sequencing primer is used for sequencing the DNA template of the RNA of interest and serves as a docking site for the T7 RNA polymerase when it is halted. Tus protein binds to Ter site and halts the transcribing RNA polymerase. Direction of transcription and sequencing are indicated by orange and purple arrows, respectively. Tus-bound Ter site that is non-permissive (halting) and permissive (read-through) to RNA polymerase are indicated by solid and open red triangles, respectively. The halted RNA transcript includes a triplet G derived from the T7 promoter, followed by the RNA of interest and some of the Illumina sequencing primer. The 3’-end of RNA transcript, indicated by a dashed line, is inaccessible. (B) Construction of DNA templates for HiTS-RAP. DNA template is constructed by PCR in two steps using 2 sets of nested oligos. Forward oligos introduce T7 promoter (step 1) and Illumina flowcell adaptor (step 2), whereas reverse oligos introduce Illumina sequencing primer (step 1), and Ter site and Illumina flowcell adaptor 2 (step 2). (C) Sequence of the HiTS-RAP DNA template for GFP aptamer. GFP aptamer encoding sequence is in green, and the rest of the sequences are colored as in (A). The transcription start site is indicated by +1 and a broken arrow. (D) Sequence of the halted RNA transcript from GFP aptamer template. Sequences are colored as in (C). Uppercase indicates the region of the halted RNA transcript that is accessible, and the lowercase indicates a region that is likely to be buried in T7 RNA polymerase and thus inaccessible by other proteins.

Article Snippet: , , Low or high DNA template concentration , Make sure the DNA template was properly quantified and diluted, as both higher and lower amounts leading to under- or over-clustering on the flowcell, respectively, can yield lower than ideal sequences in GAIIx. A 20 pM denatured DNA solution, as recommended by Illumina, can be used for cluster generation on flowcell using cBot..

Techniques: Illumina Sequencing, Binding Assay, Sequencing, Derivative Assay, Construct, Introduce

Journal: Nature protocols

Article Title: Quantitative Assessment of RNA-Protein Interactions with High Throughput Sequencing - RNA Affinity Profiling (HiTS-RAP)

doi: 10.1038/nprot.2015.074

Figure Lengend Snippet: Schematics of HiTS-RAP. (i) The HiTS-RAP DNA template, colored as in Fig. 2, is amplified on the cBot instrument to generate DNA clusters on Illumina flowcell. (ii) HiTS-RAP starts with sequencing of DNA clusters on the flowcell by the GAIIx. (iii) After sequencing, clean, full-length DNA template is regenerated, and (iv) bound by Tus protein at the Ter site. (v) DNA is then transcribed by T7 RNA polymerase, which becomes halted at the Tus-bound Ter site, retaining the RNA transcript on the DNA template. (vi) Finally, increasing concentrations of the target protein are incubated with the halted RNA transcripts, and the bound protein is monitored via the fluorescence of mOrange fusion protein. Once the assay is finished, the protein binding data is used to calculate the binding affinities that are matched with the sequence of each cluster, yielding Kd measurements for millions of RNAs per lane of the Illumina flowcell. Steps that constitute HiTS-RAP (iii-vii) are indicated with a gray colored box. Critical actions that require user input such as loading reagents and modifying file settings are indicated with a blue open triangle and italicized text.

Article Snippet: , , Low or high DNA template concentration , Make sure the DNA template was properly quantified and diluted, as both higher and lower amounts leading to under- or over-clustering on the flowcell, respectively, can yield lower than ideal sequences in GAIIx. A 20 pM denatured DNA solution, as recommended by Illumina, can be used for cluster generation on flowcell using cBot..

Techniques: Amplification, Sequencing, Incubation, Fluorescence, Protein Binding, Binding Assay

Journal: Nature protocols

Article Title: Quantitative Assessment of RNA-Protein Interactions with High Throughput Sequencing - RNA Affinity Profiling (HiTS-RAP)

doi: 10.1038/nprot.2015.074

Figure Lengend Snippet: Comparison of HiTS-RAP and RNA-MaP. Major distinguishing (highlighted with gray background) and minor technical differences between these two methods are listed. Many of the differences can be readily implemented in either method.

Article Snippet: , , Low or high DNA template concentration , Make sure the DNA template was properly quantified and diluted, as both higher and lower amounts leading to under- or over-clustering on the flowcell, respectively, can yield lower than ideal sequences in GAIIx. A 20 pM denatured DNA solution, as recommended by Illumina, can be used for cluster generation on flowcell using cBot..

Techniques: Comparison, Modification, Control, Sequencing, Protein Binding, Imaging, Extraction, Software, Fluorescence, Binding Assay, Labeling, Hybridization

Journal: Nature protocols

Article Title: Quantitative Assessment of RNA-Protein Interactions with High Throughput Sequencing - RNA Affinity Profiling (HiTS-RAP)

doi: 10.1038/nprot.2015.074

Figure Lengend Snippet:

Article Snippet: , , Low or high DNA template concentration , Make sure the DNA template was properly quantified and diluted, as both higher and lower amounts leading to under- or over-clustering on the flowcell, respectively, can yield lower than ideal sequences in GAIIx. A 20 pM denatured DNA solution, as recommended by Illumina, can be used for cluster generation on flowcell using cBot..

Techniques: Sequencing, dsDNA Assay, Concentration Assay, Real-time Polymerase Chain Reaction, Protein Binding, Modification, Functional Assay, In Vitro, Activity Assay, Binding Assay, Protein Concentration, Labeling