We followed the mononucleosome isolation protocol described in
[4 (link)]. Briefly, cells were prepared as described above for ChIP by the quenching step and resuspended in 20 ml of zymolyase buffer. Next, 250 μg of zymolyase (MP Biomedicals, Santa Ana, CA, USA, cat.# IC320921) was added to make spheroplasts, then resuspended in 2 ml NP buffer. The spheroplasts were treated with MNase (Worthington Biochemical Corp., Lakewood, NJ, USA, cat.# LS004797) at a concentration from 40 U-100 U for 10 min at 37°C. The DNA-protein complexes were reverse-crosslinked in 10 mM EDTA and 1% SDS buffer with Proteinase K at 65°C overnight. RNA was removed by RNase A treatment, then DNA was extracted with phenol-chloroform and purified by ethanol precipitation. Finally, DNA was run on an E-gel system (Invitrogen, Carlsbad, CA, USA), and approximately 147-bp DNA fragments were size-selected. Library preparation and sequencing were performed as described above for ChIP-seq.
[4 (link)]. Briefly, cells were prepared as described above for ChIP by the quenching step and resuspended in 20 ml of zymolyase buffer. Next, 250 μg of zymolyase (MP Biomedicals, Santa Ana, CA, USA, cat.# IC320921) was added to make spheroplasts, then resuspended in 2 ml NP buffer. The spheroplasts were treated with MNase (Worthington Biochemical Corp., Lakewood, NJ, USA, cat.# LS004797) at a concentration from 40 U-100 U for 10 min at 37°C. The DNA-protein complexes were reverse-crosslinked in 10 mM EDTA and 1% SDS buffer with Proteinase K at 65°C overnight. RNA was removed by RNase A treatment, then DNA was extracted with phenol-chloroform and purified by ethanol precipitation. Finally, DNA was run on an E-gel system (Invitrogen, Carlsbad, CA, USA), and approximately 147-bp DNA fragments were size-selected. Library preparation and sequencing were performed as described above for ChIP-seq.
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