D-Glucose HK Assay Kit

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

Place of Origin: Guangdong, China

Warranty: 2 years

Customized support: OEM

Brand Name: HKM

Model Number: CRM009

Reagent grade: Biochemical Reagent

Form: Powder

Color: Light yellow

Type: Vibro Cholerae Agar

1000mL

Introduction

This product is suitable for extracting total RNA from 50-150mg conventional plantor fungal samples as well as samples rich in polyphenolic and polysaccharides. This kit is based on silica gel column purification technology, and the extraction process does not require the use of toxic phenol chloroform extraction. The entire extraction process only takes 20-30 minutes. This kit adopts DNA filtration technology, which can efficiently filter and remove DNA. The obtained RNA can be directly used for experiments such as RT-PCR, Northern Blot, poly A+ purification, nucleic acid protection, and in vitro translation.

Details

Kit Contents

Contents	R415002	D415003
Purification Times	50 Preps	250 Preps
HiPure RNA Mini Columns	50	250
gDNA Filter Columns	50	250
2ml Collection Tubes	100	500
TCEP (1M)	0.29 g	5 x 0.29 g
Buffer EP	1.0 ml	5.0 ml
Buffer PSL	50 ml	250 ml
Buffer RW1	50 ml	250 ml
Buffer RW2*	20 ml	2 x 50 ml
RNase Free Water	10 ml	30 ml

Storage and Stability

Except TCEP, the kit components can be stored dry at room temperature (15–25°C) and are stable for at least 18 months under these conditions. After receiving the product, it is recommended to store TCEP (dry powder) at -20-8°C. At low temperatures, Buffer PSL may form precipitates, dissolve it completely by 55°C water bath.

Purchase Guide

1. When dealing with woody or uncommon samples, R4150 is recommended first. R4150 contains two polysaccharide/polyphenol lysis buffer, which is the most universal product.

2. R4151 is recommended for handling common economic crop samples for the first time. Strong lysis solution can be used to process easy-extraction samples. The amount of corn or rice leaves samples can reach up to 300mg.

3. R4165 adopts CTAB/chloroform method, which can also handle a large number of difficult-to-extraction plants, but requires contact with chloroform substitutes, which is less safe than other kits. This kit uses DNase Ⅰ to remove DNA, which is also a good choice for extracting polysaccharide/polyphenol-rich plant samples.

4. R4014 is recommended for fruit/starch plant samples, which uses improved trizol pre-treatment, single column operation and is more economical.

This product is suitable for extracting total RNA from 50-150mg conventional plantor fungal samples as well as samples rich in polyphenolic and polysaccharides. This kit is based on silica gel column purification technology, and the extraction process does not require the use of toxic phenol chloroform extraction. The entire extraction process only takes 20-30 minutes. This kit adopts DNA filtration technology, which can efficiently filter and remove DNA. The obtained RNA can be directly used for experiments such as RT-PCR, Northern Blot, poly A+ purification, nucleic acid protection, and in vitro translation.