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.
Our eCLIP library preparation kit is an optimized 4-day protocol based on Van Nostrand, Yeo et al. Nature Methods 2016 paper.
Why choose the eCLIP kit?
- 1000-fold improved library prep efficiency vs standard iCLIP-seq
- Increased experimental success rates, decreased wasted sequencing
- Significantly improved signal/noise ratio identifies true in vivo targets
Each kit includes:
- All eCLIP primers and buffers needed to process 8 samples (8 IP and 8 inputs)
- Size selection beads
- Quality tested transfer membrane
- Index primers
What are the sequencing parameters?
Eclipse Bio’s 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.
What is the recommended sequencing depth per sample?
Eclipse Bio’s target is 25 million reads per eCLIP-seq dataset. 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.
What genomes are available for analysis?
We have the following genomes available for analysis: human (hg38), mouse (mm10), rat (rn6), c. elegans (ce11). Custom species may incur an additional setup fee, including the addition of a viral genome to one of our available species. Contact us at firstname.lastname@example.org for more information.
What are the indices for sequencing Sample-Sheet?
|i7 index name
||i7 bases on Sample Sheet
||i5 index name
||i5 bases bases on Sample Sheet