Each eCLIP experiment uses an antibody to immunoprecipitate the RNA binding protein of interest from a biological sample (cells, tissues, or other biospecimen).

• 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.

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..

Index primer sequences: Illumina dual index primers (provided)

505: AGGCGAAG

506: TAATCTTA

507: CAGGACGTA

508: GTACTGA

705: TTCTGAAT

706: ACGAATTC

707: AGCTTCAG

708: GCGCATTA

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/

Standard analysis of eCLIP requires read adapter trimming, mapping to the appropriate genome, peak identification, and comparison against a paired input, knockout, or other suitable control sample. An example eCLIP analysis pipeline has been described by the ENCODE consortium here.

Eclipse Protocol for Crosslinking of Suspension Cell Lines

Eclipse Protocol for Crosslinking of Adherent Cell Lines

Eclipse Protocol for Crosslinking of Tissue

Contact us for more information about data analysis options and pricing.