TITLE: Chromatin Endogenous Cleavage and high-throughput sequencing (ChEC-seq) in S. cerevisiae
AUTHORS: Gabe Zentner

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.</div></div>

[STEPS]
?. [Yeast culture and harvest]
In the morning, dilute the overnight culture to OD600 = 0.2-0.3 in 50 mL YPD or SC medium in a 300 mL flask. Grow 50 mL culture at 30°C until OD600 = 0.5-0.7.
?. [Yeast culture and harvest]
The day before the experiment, inoculate 3 mL YPD or SC medium with a single colony. Grow overnight at 30°C.
?. [Yeast culture and harvest]
Harvest cells in a 50 mL conical tube at 1,500 x g for 1 min.
?. [Yeast culture and harvest]
Wash cells 3 x 1 mL Buffer A. Transfer cells to a 1.5 mL tube with the first wash and spin as above between washes.
?. [ChEC]
Permeabilize cells. Resuspend pellet in 600 µL Buffer A + 0.1% digitonin (add 30 µL 2% digitonin in DMSO to 570 µL Buffer A) and incubate at 30°C for 5 min. Remove 100 µL as zero timepoint prior to step 6.
?. [DNA extraction]
Extract nucleic acids. Add 200 µL phenol/chloroform/isoamyl alcohol, mix well, and spin at max. speed for 5 min in a microfuge. Transfer aqueous phases to new tubes, add 2 µL (10 µg) linear acrylamide and 500 µL 100% ethanol, mix, and precipitate at -80°C for ≥30 min.
?. [DNA extraction]
Add 2 µL 20 mg/mL proteinase K. Digest protein at 55°C for 20 min.
?. [DNA extraction]
Spin at max speed and 4°C for 10 min.
?. [DNA extraction]
Wash pellet with 1 mL 75% ethanol and aspirate ethanol.
?. [DNA extraction]
Briefly air-dry pellets and resuspend in 29 µL Qiagen EB or comparable buffer + 1 µL 10 mg/mL RNase A and incubate at 37°C for 10 min.
?. [DNA extraction]
Run 5 µL RNase-treated DNA on a 1.5% agarose gel to check DNA fragmentation if desired.
?. [Size selection]
Dilute RNase-treated DNA to 200 µL with Qiagen EB or comparable buffer.
?. [Size selection]
Add 160 µL Ampure beads (0.8:1 beads:sample ratio) and pipet up and down 10X to mix. Incubate at room temperature for 5 min.
?. [Size selection]
Collect beads on magnetic rack for 2 min.
?. [Size selection]
Remove the supernantant (~400 µL) to a new tube containing 16 µL 5 M NaCl (~200 mM final).
?. [Size selection]
Extract DNA from the unbound fraction. Add 400 µL phenol/chloroform/isoamyl alcohol, mix well, and spin at max. speed for 5 min in a microfuge.
?. [Size selection]
Transfer aqueous phases to new tubes, add 2 µL (10 µg) linear acrylamide and 1 mL 100% ethanol, mix, and precipitate at -80C for ≥30 min.
?. [Size selection]
Spin at max speed and 4°C for 10 min.
?. [Size selection]
Wash pellets with 1 mL 75% ethanol and remove ethanol with vacuum.
?. [Size selection]
Briefly air-dry pellets and resuspend in 25 µL Qiagen EB or comparable buffer. Recovered DNA can be quantified by Qubit and the size distribution analyzed via TapeStation using a high-sensitivity tape. Sequencing libraries can be prepared using any standard ChIP-seq-style method.
?. [ChEC]
Add 1.1 µL 1 M CaCl2 (~2 mM final), mix, and incubate at 30°C.
A recent study mapping the genome-wide distribution of components of the SAGA and TFIID coactivator complexes (PMID 31913117) with ChEC used a 10-fold lower calcium concentration with a 5 min digestion time in order to minimize background genomic digestion and overdigestion of target sites.
?. [ChEC]
At each desired time point, remove a 100 µL aliquot of the digest to a tube containing 90 µL stop solution and 10 µL 10% SDS and vortex to mix.
We have performed multiple time course ChEC experiments for both MNase-tagged factors and free MNase in distinct genetic backgrounds. For free MNase, we have observed that relatively long digestion times (≥ 2.5 min) yield cleavage of promoter nucleosome-depleted regions and nucleosome linkers due to their accessibility. It is therefore difficult to determine if enrichment of a particular factor is specific at these later time points. However, free MNase signal is quite low at short time points, allowing clear distinction between MNase-tagged factor and free MNase signal. We therefore recommend that, once an initial time course to determine the digestion kinetics of the factor under study and its corresponding free MNase control is performed, short time points be used for further experiments. We have found that a ≤ 1 min digestion is sufficient for all tested factors using the digestion conditions provided here.
?. [Data processing and analysis]
We align paired-end Illumina fastq files to the sacCer3 genome assembly with Bowtie 2 using additional parameters (-I 10 -X 700 --no-unal --no-mixed --no-discordant --dovetail). We then convert alignment SAM files to BAM format with SAMtools and use deepTools bamCoverage to generate bigWig coverage tracks with single-base resolution (-bs 1), counts per million normalization (--normalizeUsing CPM), and extension of reads to their paired-end fragment length (-e). Further analysis can be done as for ChIP-seq (e.g. peak calling using MACS2 and motif discovery with MEME or HOMER).