I have a question in Size-selection(SPRI) in ChIP-seq library preparation

Question

[oryzae51]

Actually, it is ChIPmentation protocol of "ChIPmentation CeMM v1.14 (September 2016)" but ChIP-seq library protocol of PerkinElmer, Nextflex have same step in SPRI size selection.

What I cannot understand is, after right-side size selection and transfer sup to a new tube(0.65x beads), they add 12.5μl of magnetic beads and remove sup.

I understand SPRI as "low ratio of magnetic bead only bind with large DNA, a.k.a right-side size selection". But adding 12.5μl magnetic, almost 0.15x, and removing sup step after 0.65x right-side size selection confuse me. For me, 12.5μl do not have any DNA fragment.

Is there anyone who understand this step correctly? The protocol link is ChIPmentation_CeMM, page 5, size-selection step 4. PerkinElmer, Nextflex ChIP-seq library kit has same step in size selection protocol

[jv]

this question is best directed to those who developed the protocol

Answer 1

[rpolicastro]

It's selecting against smaller fragments, so the beads will bind fragments above a certain molecular weight, and the supernatant will contain anything smaller than that. The ratios change a bit when you are adding beads to a solution that already contains the buffer the beads were stored in, so the optimal bead amount to add at this step was likely empirically determined. You can ask the kit manufacturers for more information.

Answer 2

[Carlo Yague]

It is not so much the amount of beads that direct the size selection than the proportion of bead buffer (contains ethanol, salt, etc). After you incubate with 0.65x beads, the supernatant contains smaller DNA fragments and also a significant proportion of bead buffer. Adding more beads (the 12.5 µL) to the supernant further increases the proportion of buffer in the solution and favors the binding of the smaller DNA fragments (even if the final bead volume is small).

[oryzae51]

The effect of increased proportion of buffer is quite reasonable. I overlooked that factor. Thank you for precious comment.