Description
Highlights
High Throughput and Robust Workflow
A simplified version of the Nature Methods-published eCLIP protocol enables profiling m6A marks with 40-fold less mRNA input than other approaches
Highly Reproducible with accurate data
Significant improvements in signal/noise reproducibly identifies m6A modified RNAs
Unbiased with high specificity
m6A eCLIP captures the DRACH motif and recapitulates other m6A profiling methods
The m6A-eCLIP Kit is a targeted RNA modification kit that has applications in many fields of research. Starting with purified RNA, the kit allows researchers to globally map m6A RNA modification states, which has been associated with cancer, autoimmune disease, obesity, and other disease indications.
N6-methyladenosine (m6A) is the most prevalent epitranscriptomic RNA modification found in eukaryotes, and m6A has been shown to regulate many stages of RNA biology including splicing, secondary/tertiary structure, localization, stability, and translation. Adenosine modifications are actively added, recognized, and removed by a series of “writer”, “reader”, and “eraser” proteins, making m6A a dynamic and reversible system for regulating RNAs.
The m6A-eCLIP Kit combines the use of an m6A antibody with the eCLIP technology, based on Van Nostrand et al. (Nature Methods, 2016) that allows user to accurately and robustly identify and map RBP binding sites on target RNAs. In pairing m6A and the eCLIP technology, RNA is chemically fragmented into 100 nucleotides or smaller fragments, followed by magnetic immunoprecipitation with a m6A antibody. After immunoprecipitation, isolated RNA fragments are subjected to library preparation including adapter ligation, reverse transcription, and PCR amplification. The streamlined 4-5 day protocol is easy to process and produces high quality libraries, and can be broken down into multiple start and stop steps. The kit enables the user to do mRNA isolation, m6A IP and library prep on up to 8 high quality samples.
FAQs
m6A-eCLIP Experiment
What are the sample requirements for an m6A experiment?
Our recommendation is 50ug of total RNA with a RIN score of 8 or higher.
Do you usually recommend biological replicates, and if so, how many?
We recommend running at least duplicates for publication purposes, however triplicates always produce more reliable data.
Sequencing
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 are the indices for sequencing Sample-Sheet?
| i7 index name |
i7 bases on Sample Sheet |
i5 index name |
i5 bases bases on Sample Sheet |
| 705 |
ATTCAGAA |
505 |
AGGCGAAG |
| 706 |
GAATTCGT |
506 |
TAATCTTA |
| 707 |
CTGAAGCT |
507 |
CAGGACGT |
| 708 |
TAATGCGC |
508 |
GTACTGAC |
If I have a project that needs additional index primers what primers are compatible?
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).
What are the adapter sequences?
RNA adapter: 5Phos/rArGrArUrCrGrGrArArGrArGrCrArCrArCrGrUrCrUrG/3SpC3/
ssDNA adapter: 5Phos/NNNNNNNNNNAGATCGGAAGAGCGTCGTGT/3SpC3/
What are the index primer sequences?
Index primer sequences: Illumina dual index primers (provided)
505: AGGCGAAG
506: TAATCTTA
507: CAGGACGT
508: GTACTGAC
705: TTCTGAAT
706: ACGAATTC
707: AGCTTCAG
708: GCGCATTA
