Bisulfite seq is a well know technology to detect DNA methylation and several technologies such as WGBS, RRBS, MeDIP-Seq, and MSBS are used for whole genome DNA methylation analysis. DNA methylation is important for regulation of cell development, differentiation and gene expression in molecular biology, genetics and epigenetics. Most methylated cytosines are found at CpG sites, and 70-80% of cytosines are methylated. The number of CpG sites in human genome is around 28 million, which is less than 1% of the genome compared with 4.4% expected.

Whole genome bisulfite sequencing (WGBS) is the most effective method of DNA methylation analysis. The only limitation is the sequencing cost is very high because the whole genome is sequenced including all the non-methylated regions.

Reduced Representation Bisulfite Sequencing (RRBS) is the reduced representation of a smaller fraction of the methylated CpG sites. RRBS combines restriction enzyme digestion and bisulfite sequencing, and enriches the sequencing for methylated CpG sites. It is an efficient technology for estimate the whole genome methylation patterns at the single base level. Although this allows a higher coverage depth and reduces the sequencing cost, the limitation is only 10% of the methylated CpG sites are covered.

Methylated DNA Immunoprecipitation Sequencing (MeDIP-Seq) is another whole genome enrichment technique used for selection of methylated DNA. Using antibodies against 5-methylcytosine, methylated DNA is enriched from whole genomic DNA via immunoprecipitation. 5-methylcytosine antibodies are incubated with fragmented genomic DNA and precipitated, followed by DNA purification and sequencing. There are several drawbacks of MeDIP-Seq: 1. Low resolution (150~200 bp) as opposed to the single base resolution; 2. Non-specific interaction due to antibody specificity and selectivity. 3. Bias towards hypermethylated regions.

The Methylation Specific Bisulfite Seq (MSBS) Library Prep Kit (illumina platform) was developed for construction of NGS libraries for methylated CpG sites using bisulfite treated DNA (20 ng – 500 ng) as input. The kit enriches methylated CpG regions, thus significantly reduce the sequencing cost. The kit estimates the whole genome methylation patterns at the single base level since it is based on a bisulfite-seq technology.

It is known that bisulfite treatment of completed NGS libraries causes tremendous damage to the libraries. By using bisulfite treated DNA as input, the kit overcomes the significant library loss due to the bisulfite conversion. The kit contains a mixture of PCR polymerases that have high-fidelity amplification and uracil tolerance which is ideal for bisulfite treated DNA.

Methylation Specific Bisulfite Seq Library Prep Kit Workflow

Three index types are available for the kit:

Non-index (Cat.# 30101): Libraries do not have index.

Index (Cat.# 30102): Each primer contains a unique barcode sequence of 6 bases to identify the individual library. Library multiplexing capacity is up to 48 samples. Index information can be downloaded here.

Unique dual index (Cat.# 30103): The multiplexing of bisulfite sequencing library is up to 96 samples with unique dual indexes. We used a Four-Base Difference Index System to generate indexes that have at least 4 bases different from each other in the 8-base index. The index primers remove NGS errors including index cross-contamination, index hopping, reads mis-assignment etc. Index information can be downloaded here.

Methylation Specific Bisulfite Seq advantages

• • • Enrichment of methylated CpG sites
    • Single-base resolution
    • Low cost for sequencing
    • Fast
      • Total time: 1.5 hours
      • Hands-on time: 10 minutes
    • Simple workflow
    • Bisulfite treated DNA as input: From 20 ng to 500 ng

MSBS Library Prep Kit enriches CpG sites

High methylation regions and low methylation regions in human genome.

High methylation region in human genome.

Low methylation region in human genome.

Sequencing setting: Single-end 35 cycles (Read 1, 35 bases) recommended
To maximize the methylated CpG enrichment, we recommend to sequence the MSBS libraries with single end 35 cycles (read1, 35 bases). This is because the enriched methylated CpG sites are mainly located around the beginning of read 1 sequences. Shorter single end reads tend to have better methylated CpG enrichment.

Bisulfite seq is a well know technology to detect DNA methylation and several technologies such as WGBS, RRBS, MeDIP-Seq, and MSBS are used for whole genome DNA methylation analysis. DNA methylation is important for regulation of cell development, differentiation and gene expression in molecular biology, genetics and epigenetics. Most methylated cytosines are found at CpG sites, and 70-80% of cytosines are methylated. The number of CpG sites in human genome is around 28 million, which is less than 1% of the genome compared with 4.4% expected.

Introduction

This kit is used for extracting total viral nucleic acid from non-cell/low cell content biological samples such as body fluid, serum, plasma, urine, immersion solution, tissue homogenate supernatant, culture supernatant, etc., the extracted products can be used for clinical in vitro detection.

Details

Specifications

Principle

This product is based on silica gel purification. The sample is lysed and digested with lysate and protease, DNA / RNA is released into the lysate. Transfer to an adsorption plate and filter column. DNA/RNA is adsorbed on the membrane, while protein is not adsorbed and is removed with filtration. After washing proteins and other impurities, DNA / RNA was finally eluted with low-salt buffer (10 Mm Tris, pH 8.0).

Advantages

• Fast – several samples can be extracted in 20 minutes by column method
• High quality – high purity total RNA can be directly used in various sensitive downstream applications
• Safe – no phenol chloroform extraction required
• Sensitive – DNA/RNA can be recovered at the level of PG
• High yield – carrier RNA contained in the product maximize the recovery of trace nucleic acid

Kit Contents

Storage and Stability

Proteinase K should be stored at 2–8°C upon arrival. However, short-term storage (Proteinase K up to 8 weeks) at room temperature (15–25°C) does not affect their performance. The remaining kit components can be stored at room temperature (15–25°C) and are stable for 18 months under these conditions.

Experiment Data

This kit is used for extracting total viral nucleic acid from non-cell/low cell content biological samples such as body fluid, serum, plasma, urine, immersion solution, tissue homogenate supernatant, culture supernatant, etc., the extracted products can be used for clinical in vitro detection.

Overview

• Ensure optimal results for sensitive applications like NGS
• Up to a tenfold increase in microRNA mapping during sequencing runs to reduce costs
• The purified exosomal RNA is free from any circulating RNA-binding proteins.
• No phenol extractions, Proteinase K treatment, nor carrier RNA.
• No time-consuming ultracentrifugation, filtration nor special syringes are required.
• Concentrate isolated RNA into a flexible elution volume ranging from 50 μL to 100 μL.
• Purification is based on EXTRAClean spin column chromatography that uses Norgen’s proprietary separation matrix.

Norgen’s EXTRAClean Plasma/Serum Exosome and Free-Circulating RNA Isolation Kit constitutes an all-in-one system for the purification of exosomes and the sequential isolation of RNA and free-circulating RNA from different plasma/serum sample volumes ranging from 50 μL and up to 10 mL. The purification is based on spin column chromatography that employs Norgen’s proprietary resin. The EXTRAClean columns undergo stringent processing and rigorous quality control measures to minimize contamination traces, ensuring optimal results for sensitive applications such as NGS. The kit is designed to isolate all sizes of RNA, including microRNA. The kit provides a clear advantage over other available kits in that they do not require any special instrumentation, protein precipitation reagents, extension tubes, phenol/chloroform or any protease treatments. Moreover, the kit allows the user to elute into a flexible elution volume ranging from 50 μL to 100 μL. The RNA isolated from the purified exosomes is free from any protein-bound circulating RNA and is of the highest integrity. Moreover, the free-circulating, protein-bound, RNA is free from any exosomal RNA. The purified RNA can be used in a number of downstream applications including real time PCR, NGS application, reverse transcription PCR, Northern blotting, RNase protection and primer extension, and expression array assays.

Details

Supporting Data

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Storage Conditions and Product Stability
All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years after the date of shipment. It is recommended to warm Lysis Buffer A for 20 minutes at 60°C if any salt precipitation is observed.