Plant Protein-RNA Interaction Binding Site Assay Service

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Plant Protein-RNA Interaction Binding Site Assay Service

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Post-transcriptional gene regulation is essential in plant biological processes. The underlying molecular mechanism is mediated by the interaction of RNA transcripts with many RNA binding proteins (RBPs) at the physical level, which regulate the properties of RNA splicing, stability, nuclear export, and translation. Therefore, CD BioSciences helps clients to elucidate the RNA binding preferences of these RBPs and map the proteomes, which helps to unravel the biological mechanisms of RNA-protein interactions.

Introduction of CLIP-seq and LACE-seq

CLIP-seq is a second-generation sequencing (NGS) based method for mapping RNA binding at interacting proteins or RNA modification sites on a genome-wide scale.

Overview of the general CLIP workflow Figure 1. Overview of the general CLIP workflow. (Hafner, M., et al., 2021)

LACE-seq is a sequencing method for researching the genome-wide binding sites of RNA-binding proteins and can be used to characterize the mechanism of RBP function.

Schematic of the LACE-seq method. Figure 2. Schematic of the LACE-seq method. (Su, R., et al., 2021)

Service Content

CLIP-seq service

We provide one-stop CLIP-seq services to help clients explore precise RNA-protein interaction sites and more gene regulatory functions.

  • Service flow

1) RBP proteins are covalently bound to RNA by UV irradiation or enzymatic reactions.

2) RNA-protein complexes are isolated by immunoprecipitation with specific antibodies after cell lysis.

3) The RNA is sheared and ligated to the junction.

4) Remove impurity proteins and sequence RBPS binding sites using RNAseq technology.

  • Service expansion
Service type Details
HITS-CLIP Introduced the ability to add dinucleotide barcodes to primers, providing the ability to sequence and deconvolve multiple experiments simultaneously. By analyzing crosslink-induced mutation sites (CIMS) at high sequencing depth, crosslinked sites can be distinguished from other sources of sequence variation.
PAR-CLIP It can identify binding sites for cellular RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs).
iCLIP It amplifies truncated cDNAs that are produced when reverse transcription stops prematurely at the cross-linking site.
eCLIP It improves iCLIP by increasing the efficiency of converting purified RNA fragments into cDNA libraries, enabling rigorous quantitative standardization against paired input controls and quantitative comparisons between peaks and samples.

LACE-seq service

Our LACE-seq technology identifies RBP targets of action in microcells and enables precise identification of RBP binding sites at single-base resolution and single-cell level by linear amplification of the termination signals of reverse transcriptase at RBP binding sites.

  • Service flow

1) Cross-linking of cells with UV-C light.

2) Dephosphorylation of the 3' end of the fragmented RNA and ligation to the 5' pre-adenylate linker.

3) Reverse transcription with biotinylated primers containing the T7 promoter.

4) The cDNA terminating at the RBP-RNA cross-linking site is poly(A)-tailed and enriched for PCR amplification and in vitro transcription.

5) Linearly amplified RNA was transformed into libraries for single-end deep sequencing.

Notification for Sample Preparation and Handling

Type Details
Sample requirements RNA ≥ 50ug (Please ensure the RNA is not visibly degraded.)
Sample storage RNA can be dissolved in ethanol or RNA-free ultrapure water and stored at -80°C. RNA should avoid repeated freezing and thawing.
Transportation Store RNA samples in 1.5 mL Ep tubes sealed with the airtight film. It is recommended that 5-10 pounds of dry ice be transported every 24 hours.

Deliverables

FastQ, BAM, coverage summary, QC reports, and customized bioinformatics analysis.

At CD BioSciences, we offer a range of protein-nucleic acid interaction analysis services. We can accurately characterize RBP's RNA-binding sites at single-base resolution and single-cell level in microcells with high-quality and reliable results. Contact us now to learn more about our services which will deepen your understanding of post-transcriptional regulatory networks and mechanisms.

References

  1. Hafner, M., et al. (2021). CLIP and complementary methods. Nat Rev Methods Primers. 1, 20.
  2. Su, R., et al. (2021). Global profiling of RNA-binding protein target sites by LACE-seq. Nature Cell Biology. 23(6), 664-675.

For research use only, not for clinical use.