RRBS NGS library prep

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I am trying to prepare RRBS sequencing libraries with the NEBNext® DNA Library Prep Master Mix Set for Illumina® kit and NEBNext® Multiplex Oligos for Illumina® (Methylated Adaptor) kit. I used 5 ug starting DNA for each sample. However, I encounter several problems and I hope to find answers here.
1. I used AMPure XP beads for sample clean up as suggested by the NEB protocol. Generally, the recovery rate for each clean up was above 85% except for the step after dA-tailing of end-repair product. The recovery rate was always below 45%. I repeated this step several times and the yield was the same. Is there something in the buffer or dA-tailing enzyme that inhibits bead binding efficiency? Can I improve the yield by adding more beads than 1.8X as suggested by the NEB DNA Library Prep protocol?

2. The RRBS protocol that comes with the NEBNext® Multiplex Oligos for Illumina does not select proper size of DNA after adaptor ligation. I tried the dual bead-based size selection as suggested in the NEB DNA Library Prep protocol, but I am not able to recover any DNA with insert size of 100 or 200 bp. Does anyone have a working bead-based size selection protocol for RRBS? Does gel selection (3% Nusieve 3-1 agarose gel) or bead selection work better?

Thank you and I look forward to your answers!

[reprogrammer]

I meet the same issue, have you figured out how to solve it, please?

[jdk787]

I don't know what is happening with your post A-tailing cleanup, but when size selecting for RRBS you need to keep in mind that only ~1% of your sample will be in the 220-320bp (100-200bp insert) range so a very low recovery rate is expected.

In my experience using beads produces a better yield than gel selecting, but both will work.

[reprogrammer]

I don't know what is happening with your post A-tailing cleanup, but when size selecting for RRBS you need to keep in mind that only ~1% of your sample will be in the 220-320bp (100-200bp insert) range so a very low recovery rate is expected.

In my experience using beads produces a better yield than gel selecting, but both will work.

I don' t understand how do you use beads instead of gel selecting, do you mean to use beads to purify the selected gel fragment but not use the column in the kit?

[jdk787]

I don' t understand how do you use beads instead of gel selecting, do you mean to use beads to purify the selected gel fragment but not use the column in the kit?

I mean using only beads to perform the size-selection instead of using a gel at all.

This blog post explains the process of size selection with beads pretty well.

http://core-genomics.blogspot.com/2012/04/how-do-spri-beads-work.html