EpiTect Bisulfite Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	For best results, the CT Conversion Reagent should be prepared freshly and used immediately following preparation.

Protocol tips
For best results, the CT Conversion Reagent should be prepared freshly and used immediately following preparation.

Publication protocol

Identification of methylated cytosines
At each reference cytosine, the binomial distribution was used to identify whether at least a subset of the genomes within the sample were methylated, using a 0.01 FDR-corrected P value. We identified methyl cytosines while keeping the number of false-positive methylcytosine calls below 1% of the total number of methyl cytosines we identified. The probability P in the binomial distribution B(n, P) was estimated from the number of cytosine bases sequenced in reference cytosine positions in the unmethylated Lambda genome (referred to as the error rate: nonconversion plus sequencing error frequency). We interrogated the sequenced bases at each reference cytosine position one at a time, where read depth refers to the number of reads covering that position. For each position, the number of trials (n) in the binomial distribution was the read depth. For each possible value of n we calculated the number of cytosines sequenced (k) at which the probability of sequencing k cytosines out of n trials with an error rate of p was less than the value M, where M* (number of unmethylated cytosines) <0.01* (number of methylated cytosines) and if the error rate of p was over 0.01, we assumed that the cytosine was not methylated. In this way, we established the minimum threshold number of cytosines sequenced at each reference cytosine position at which the position could be called as methylated, so that out of all methyl cytosines identified no more than 1% would be because of the error rate.

Calculation of DNA methylation level
If the error rate is less than 0.01 we calculated adjusted DNA methylation level for cytosine as follow:

An external file that holds a picture, illustration, etc.
Object name is ncomms6619-m1.jpg
(a=total Cs, b=number of converted Cs, cr=bisulfite conversion rate).

Identification of DMRs
DMRs (Fig. 2) were identified using a sliding window approach (Supplementary Fig. 6a, Fig. 2b). A window size of 30 CpGs less than 6 kb with coverage more than 5 × in 15 CpGs per window in all samples were considered, progressing one CpG per iteration. Total of 20,214,978 windows were assessed. Windows showing maximum difference and fold enrichment of 30% and fourfold with Benjamini–Hochberg-corrected FDR from analysis of variance (ANOVA) test P values of less than 1% were identified as differentially methylated windows. In all, 188,529 differentially methylated windows were then joined if regions were overlapped or progressing region and the succeeding regions were covering more than 60% of the region. This set of 7,890 DMRs covering 21,618,964 bp of the whole genome are reported in Fig. 2 and Supplementary Data 2.

DMRs were then defined as Hyper-DMRs and Hypo-DMRs if the average methylation level difference of each DMR in each sample was higher or lower by more than 20% relative to 2°MEF.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing DNA methylation profiling Whole genome profiling - mouse iPSCs using EpiTect Bisulfite Kit from Qiagen.

Manufacturer protocol

Download the product protocol from Qiagen for EpiTect Bisulfite Kit below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing DNA methylation profiling Whole genome profiling - mouse iPSCs using EpiTect Bisulfite Kit from Qiagen. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.