Rapid and inexpensive preparation of genome-wide nucleosome footprints from model and non-model organisms

Abstract

MNase-seq (micrococcal nuclease sequencing) is used to map nucleosome positions in eukaryotic genomes to study the relationship between chromatin structure and DNA-dependent processes. Current protocols require at least two days to isolate nucleosome-protected DNA fragments. We have developed a streamlined protocol for S. cerevisiae and other fungi which takes only three hours. Modified protocols were developed for wild fungi and mammalian cells. This method for rapidly producing sequencing-ready nucleosome footprints from several organisms makes MNase-seq faster and easier, with less chemical waste.

Keywords: Genomics; Model Organisms; Molecular Biology; Sequencing.

Graphical abstract

Figure 1

Sample gel showing properly digested nucleosome footprints Agarose gel showing nucleosome footprints recovered from liquid culture of S. cerevisiae using the rapid MNase protocol. (A) and (B) are replicates from the same WT yeast strain.

Figure 2

Rapid MNase can accurately map nucleosome positions in S. cerevisiae cells (A) Nucleosome dyad signal at 4655 yeast transcription start sites (TSSs) are plotted for WT and isw2 nucleosomes harvested using a standard or rapid protocol. (B) Example Genome Browser image showing the standard method and rapid method can map Isw2-directed nucleosome positions similar to published data sets at the RAD51 locus. Dashed lines indicate Isw2-positioned nucleosomes. (C) Nucleosome dyad signal at 202 intergenic Ume6 target sites showing rapid and standard MNase methods can accurately identify global changes in nucleosome structure at an Isw2 recruitment motif. (D) Genome Browser image showing all methods correctly identify Isw2-positioned nucleosomes at the MEI4-ACA1 locus, a Ume6 target site. Dashed lines indicate Ume6- and Isw2- positioned nucleosomes.

Figure 3

Rapid MNase can recover nucleosome footprints from isolated quiescent cells and yeast patches (A) Representative gel showing nucleosome footprints recovered from purified quiescent cells using the rapid MNase protocol. (B) Representative gel (right) showing nucleosome footprints recovered from a fresh patch of yeast collected from YPD-Agar (left). (C) Nucleosome dyad signal at transcription start sites (TSSs) comparing standard and patch- recovered “colony” MNase footprints. (D) Example Genome Browser image showing “colony” MNase footprints can accurately identify Isw2-directed nucleosome positions at the ESC8 locus compared to the standard MNase protocol and published data sets. (E) Nucleosome dyad signal at 202 intergenic Ume6 target sites showing “colony” MNase can accurately identify global changes at an Isw2 recruitment motif. (F) Genome Browser image showing colony MNase can similarly identify Isw2-positioned nucleosomes at the YIG1-CSM4 locus, an example Ume6 target site. Dashed lines indicate Ume6- and Isw2-positioned nucleosomes.

Figure 4

Rapid MNase can accurately map nucleosome positions in S. pombe and N. crassa (A) Agarose gel showing example nucleosome footprints recovered from S. pombe using the rapid MNase protocol. (B) Genome Browser image comparing nucleosome dyad positions on S. pombe chrII recovered for the rapid MNase protocol (top) and previously- published data sets. (C) Alignment of nucleosome dyads at 11,350 transcription start sites (TSSs) for rapid MNase-recovered nucleosome footprints and previously-published data sets. (D) Agarose gel showing example nucleosome footprints recovered from N. crassa using the rapid MNase protocol. (E) Genome Browser image comparing nucleosome dyad positions at the N. crassa NCU3995-NCU3994 locus for the rapid MNase protocol (top) and previously-published data sets.

Figure 5

Nucleosome footprints can be rapidly recovered from wild mushroom samples (A) Images of locally-foraged wild mushrooms that were subjected to the rapid MNase protocol (top). Sample 5-6 consists of a distinct surface fungal specimen (5) growing on a host specimen (6). Recovered nucleosome footprints for corresponding mushrooms are shown (bottom). Speculative identities for these samples are (1) Panaeolus foenisceii, (2) unknown, (3) Craterellus tubaeformis, (4) Cantharellus formosus, (5) Hypomyces lactifluorum, (6) Russula brevines, (7) Lycoperdon perlatum. (B) Optimized rapid MNase for specimen 4 (chanterelle) was achieved using 50 mg of tissue leading to well-digested nucleosome footprints (top). The optimized protocol was performed on 50 mg of a previously-untested specimen leading to well-digested nucleosome footprints (bottom). Speculative identity of these samples are Cantharellus formosus (top) and Agricus xanthodermus (bottom).

Figure 6

The rapid MNase protocol can be performed on human cells with or without crosslinking (A) Cartoon schematic showing rapid MNase protocol and associated nucleosome footprints for 1 million human cells from the diploid myeloid leukemia cell line PLB-895. The protocol is identical to the yeast liquid culture protocol except that there is no zymolyase treatment. (B) Cartoon schematic showing rapid MNase protocol for PLB-895 cells and associated nucleosome footprints when the crosslinking and crosslinking reversal steps are omitted and other steps are shortened. The crosslink-free protocol can provide nucleosome footprints that are ready for library construction in less than an hour. Left lane: 1.5 million cells input; right lane: 1 million cells input.

Figure 7

Sample gel showing intact genomic DNA and partial nucleosome footprints from non-permeabilized cells Agarose gel showing example nucleosome footprints recovered from patches of S. cerevisiae using unoptimized “rapid colony MNase” protocol (2 mg of zymolyase for 15 mi) using an input of (A) 30 OD pellet of yeast, or (B) 20 OD pellet of yeast.

Figure 8

Sample gel showing under- and over-digested nucleosome footprints Agarose gel showing example nucleosome footprints recovered from S. cerevisiae liquid culture using the rapid MNase protocol (A) Only 15 OD of cells were used as input. (B) Under-digested sample. (C) Under-digested sample. (D) Over-digested sample. (E) Under-digested sample. (F) Over-digested sample.