The eCLIP kit is a simplified and robust library preparation method to identify your RNA-binding proteins targets.
Eliminate time-consuming prep work by ordering one kit with 30+ qualified reagents. Start immediately and generate eCLIP libraries in only 4 days.
Published method and protocol improvements increase workflow efficiency and identify true binding sites with single nucleotide resolution
Kits include validated protocol and qualified reagents to ensure reproducibility for better results.
You can also add data analysis to your eCLIP kit library preparation kit
Our eCLIP library preparation kit is an optimized 4-day protocol based on Van Nostrand, Yeo et al. Nature Methods 2016 paper.
Based on Van Nostrand, Yeo et al. Nature Methods 2016 paper
Why choose the eCLIP kit?
Each kit includes:
$1,993.00 – $2,695.00
Simplified and robust identification of your RNA-binding protein targets in one week
|Add Data Analysis||
eCLIP Kit Protocol (v1.01R)
- Pre-validated antibodies are available for 150 RNA binding proteins; alternatively, we recommend antibody validation be performed prior to eCLIP using the eCLIP immunoprecipitation validation kit.
- Biosamples should be UV cross-linked according to protocols available for Suspension Cells and Adherent Cells.
Eclipse Bio’s eCLIP kit is based on the single-end eCLIP variant described in:
Van Nostrand EL, Nguyen TB, et al. Robust, Cost-Effective Profiling of RNA Binding Protein Targets with Single-end Enhanced Crosslinking and Immunoprecipitation (seCLIP). Methods Mol Biol. 2017;1648:177-200. PMID: 28766298.
Libraries generated using the eCLIP-seq method are typically sequenced using standard SE50 or SE75 conditions on the Illumina HiSeq, NovaSeq, or NextSeq platforms. eCLIP-seq libraries are compatible with paired-end sequencing if desired by the user, however due to the small size of typical eCLIP RNA fragments (~200bp), most fragments are fully sequenced in standard single-end formats.
How deeply to sequence an eCLIP-seq dataset is a challenging balance between cost and sufficient read depth to detect true binding events. In an effort to experimentally address this question, an analysis of eCLIP-seq datasets for 150 RNA binding proteins suggested that for 90% of datasets, saturation of peak information occurred at or below 8.5 million reads (See Supplementary Fig. 11 of Van Nostrand EL, et al. A Large-Scale Binding and Functional Map of Human RNA Binding Proteins. Nature (Accepted, in press) However, we have found that targeting 25 million reads provides better coverage for abundant, broadly binding RNA binding proteins (such as HNRNPs) while still allowing pooling of ~14 eCLIP libraries per standard Illumina HiSeq 4000 lane..
Index primer sequences: Illumina dual index primers (provided)
We recommend using NEBNext HT (cat # E7600S) index primers for larger projects. Use 5ul of both the forward and reverse primer (10uM each) then scale the total master mix volume up to 50ul (5ul Forward primer, 5ul Reverse primer, 15ul cDNA and 25ul Primer Mix).
RNA adapter: 5Phos/rArGrArUrCrGrGrArArGrArGrCrArCrArCrGrUrCrUrG/3SpC3/
ssDNA adapter: 5Phos/NNNNNNNNNNAGATCGGAAGAGCGTCGTGT/3SpC3/