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!
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!
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