Di Hu
posted 9 months ago
neuroscientist studying longevity | currently working on: single cell (epi)genomics methods development; epigenetic reprogramming; single cell multiomic human brain atlas

SPRI bead size selection guidance for histone marks

You've prepared your epigenetic library for profiling histone marks. If you are not using the Zymo ChIP DNA Clean & Concentrator Kit for final elution, you may want to size select for your library region of interest using SPRI bead clean up. How much bead do you add relative to your volume of library?

Because adapters for histone mark profiling (e.g. in ChIP-seq, CUT&Tag, TIP-seq) are 150bp and DNA wrapped around a mononucleosome is ~150bp (~300bp total for a mononucleosome), I found it ideal to size select for >200bp = 0.85X bead clean e.g. using Agencourt AMPure XP, Beckman Coulter, A63880

This way you also rid of adapter dimers in 1 step minimising sample loss.

Example protocol: library after final PCR = 40 µl

  1. Add 34 µl (40 µl * 0.85X) SPRI bead at room temperature (or warm to 26°C if your lab is cold), mix well
  2. Incubate at 26°C for 10 minutes
  3. Place on magnetic rack for 3-5 minutes until supernatant is completely clear
  4. Discard supernatant and wash twice with 200 µl freshly prepared 80% ethanol
  5. Elute with 20-30 µl water or 10 mM Tris-HCl pH 8 - incubate with beads for 5-10 minutes at 26°C
  6. Collect final library without any beads and proceed to Qubit and Bioanalyser
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