Hi, I'm doing bisulfite sequencing to study DNA methylation status of my target gene.

I'm using QIAamp DNA Mini kit(QIAGEN) for obtaining gDNA samples and EZ DNA Methylation-Lightning Kit(Zymo Research) for bisulfite conversion.

As protocol from zymo said that 200~500ng gDNA 20ul is optimal condition for bisulfite conversion, I'm using 200~500ng gDNA(sometime 500ng if my gDNA concentration is enough, but sometime I use around 200~300ng if my gDNA concentration is low.)

My problem is I think I'm getting unexpected results.

In my lab I'm the first person to study DNA methylation and use such kind of kits, I'm trying to get same, or similar results from previously published paper before I get results of my target gene.

The gene used in the paper was highly methylated and methylation status of each CpG sites were around 60~90%(mostly 70, 80%)

But when I did sequencing, only 50% were methylated.

Also CpG sites from each colony was all methylated or all unmethylated.

(I mean, I tried sequencing from 8 colonies and results from 4 colonies said all of the CpG was methylated and results from rest of the colonies said all of the CpG was unmethylated-so I'm hard to trust my results.)

I don't think bisulfite reaction didn't work because almost of non-CpG cytosines was converted to uracil.

I think I'm doing something wrong with my expreiments, so I needs some advices.

1. Is gDNA around 200~300ng not sufficient to perform bisulfite conversion? I've heard that too less gDNA amount or too much bisulfite treating could cause conversion of methylated cytosin to uracil. If it's not sufficient, how much would be optimal?

2. Is it common to get only all-or-none methylated result as me?

3. I didn't use RNaseA during extracting gDNA so maybe some RNA will be remaining in my gDNA. Would this be big problem?

4. Are there some other factors that could be a cause of unsuccessful or unreliable bisulfite conversion/sequencing? (There would be a lot, but I want to check if I'm missing something.)

If you have any other ideas or advices to perform successful bisulfite sequencing, I would like to know it whatever it is. Thank you for reading long question.