We recommend using a newer version of this product: EpiNext 5-mC RNA Bisulfite-Seq Easy Kit (Illumina) (Cat. #P-9007)
The EpiNext™ RNA Bisulfite-Seq Kit (Illumina) is a complete set of optimized reagents designed to carry out RNA bisulfite conversion, followed by a "post-bisulfite" library preparation process for Illumina platform-based bisulfite sequencing, all in one kit. Intended applications include whole transcriptome RNA bisulfite sequencing and various other RNA bisulfite-based next generation sequencing techniques for RNA methylation analysis. The optimized protocol and components of the kit allow the RNA to be bisulfite converted and fragmented simultaneously followed by quick non-barcoded (singleplexed) and barcoded (multiplexed) library construction using low-nanogram quantities of bisulfite converted RNA. The kit has the following advantages:
- Fast and streamlined procedure: The entire procedure can be finished in 6 hours. Gel-free size selection/purification saves time and prevents handling errors, as well as loss of valuable samples.
- Complete conversion: The innovative reagent composition converts unmethylated cytosine into uracil at a level greater than 99.9%, with no or negligible inappropriate/error conversion of methylcytosine to thymine (<0.1%) when the indicated range of sample RNA is used.
- High sensitivity, efficiency and flexibility: Can be used for both non-barcoded (singleplexed) and barcoded (multiplexed) library preparation. Optimized RNA bisulfite method and enhanced adaptor ligation eliminates loss of fragments and selection bias, which enables input RNA to be as low as 5 ng.
- Extremely convenient: The kit contains all the required components for each step of the RNA library preparation process, which is sufficient for bisulfite conversion, ligation, clean-up, size selection, and library amplification, thereby allowing the bisulfite RNA library preparation to be streamlined for the most reliable and consistent results.
- Minimal bias: Ultra HiFi amplification enables achievement of reproducibly high yields of bisulfite converted RNA libraries with minimal sequence bias and low error rates.
It has been observed that in humans, 5-methylcytosine occurs in various RNA molecules including tRNAs, rRNAs, mRNAs and non-coding RNAs (ncRNAs). At least 10,275 5-mC candidate sites were discovered in mRNAs and ncRNAs, which cover 10.6% of the total cytosine residues in the transcriptome. 5-mC seems to be enriched in some classes of ncRNA, but relatively depleted in mRNAs. However, the majority (83%) of their candidate sites were found in mRNAs. Within these transcripts 5-mC appears to be depleted within protein coding sequences but enriched in 5’ and 3’ UTRs. Two different methyltransferases, NSUN2 and DNMT2 are known to catalyze 5-mC modification in eukaryotic RNA. Recent data strongly suggest that RNA cytosine methylation affects the regulation of various biological processes such as RNA stability and mRNA translation. Furthermore, loss of 5-mC in vault RNAs causes aberrant processing into Argonaute-associated small RNA fragments that can function as microRNAs. Thus, impaired processing of vault ncRNA may contribute to the etiology of human disorders related to NSun2-deficiency. Bisulfite conversion of RNA followed by next generation sequencing yields reliable information about RNA cytosine methylation states on a transcriptome-wide scale.
Principle & Procedure
This kit includes all reagents required for a successful RNA bisulfite conversion and bisulfite RNA library preparation using bisulfite-converted RNA generated from a wide range of input RNA amounts (5 ng to 1 µg). In this preparation, RNA is simultaneously bisulfite converted and fragmented to the appropriate length during the bisulfite process. cDNA is synthesized from the bisulfite-treated RNA and used for ligation with specific adaptors that are necessary for amplification and sequencing. The fragments are size selected and purified using MQ binding beads, which allows for quick and precise size selection of DNA. Size-selected DNA fragments are amplified using a high-fidelity PCR Mix, which ensures maximum yields from minimum amounts of starting material and provides highly accurate amplification of library DNA with low error rates and minimal bias.
Starting materials can be total RNA isolated from various tissue/cell samples such as fresh and frozen tissues, cultured cells from a flask or microplate, microdissection samples, and body fluid samples, etc. The amount of RNA for each bisulfite reaction can be 5 ng to 1 µg. For an optimal reaction, the input RNA amount should be 200 to 500 ng. The yield of RNA purified after bisulfite conversion depends on the amount of input RNA, nature of RNA, and source of the starting material.
Fig. 1. Workflow of the EpiNext™ RNA Bisulfite-Seq Kit (Illumina).
Fig. 2. Size distribution of library fragments: Post-bisulfite cDNA library was prepared from 10 ng of input RNA using the EpiNext™ RNA Bisulfite-Seq Kit (Illumina).