TITLE: An improved ChEC-seq method for mapping the genome-wide binding of S. cerevisiae transcription factors
AUTHORS: Rafal Donczew, Amélia Lalou, Steven Hahn

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">ChEC-seq and other nuclease-based methods such as Cut&Run map protein locations on DNA by targeting nuclease activity to specific transcription factors and mapping the resulting DNA cleavages (Schmid et al. 2004; Skene and Henikoff 2017; Zentner et al. 2015). For ChEC-seq, yeast cells expressing a protein-micrococcal nuclease (MNase) fusion are permeabilized, MNase is activated by the addition of calcium, and the resulting DNA fragments are mapped. Potential advantages of this approach include avoiding non-specific protein-DNA crosslinking in highly transcribed regions, efficient mapping of factors that do not directly bind DNA and, more sensitive detection of protein-DNA interactions. We optimized the original ChEC-seq protocol to minimize non-specific DNA cleavage, avoid over digestion at authentic binding sites, and efficiently assay large numbers of factors. We also created a robust data analysis pipeline that incorporates peak calling to map binding sites and quantitative analysis, based on utilization of spike-in DNA, to compare factor-DNA binding under different conditions. We used this modified approach to map genome-wide distributions of the transcription coactivators TFIID and SAGA (Donczew et al. 2020) as well as transcription factors Abf1 and Rap1 (Donczew et al, submitted to Mol Cell).</div><div class = "text-block"><span style = "font-weight:bold;">References</span></div><div class = "text-block"><span>Donczew R, Warfield L, Pacheco D, Erijman A, Hahn S. 2020. Two roles for the yeast transcription coactivator SAGA and a set of genes redundantly regulated by TFIID and SAGA.</span><span style = "font-style:italic;">eLife </span><span>9: e50109.</span></div><div class = "text-block"><span>Schmid M, Durussel T, Laemmli UK. 2004. ChIC and ChEC; genomic mapping of chromatin proteins.</span><span style = "font-style:italic;">Mol Cell </span><span>16: 147–157.</span></div><div class = "text-block"><span>Skene PJ, Henikoff S. 2017. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites.</span><span style = "font-style:italic;">eLife </span><span>6: e21856.</span></div><div class = "text-block"><span>Warfield L, Ramachandran S, Baptista T, Devys D, Tora L, Hahn S. 2017. Transcription of nearly all yeast RNA Polymerase II-transcribed genes is dependent on transcription factor TFIID.</span><span style = "font-style:italic;">Mol Cell</span><span>. 68:118-129</span></div><div class = "text-block"><span>Zentner GE, Kasinathan S, Xin B, Rohs R, Henikoff S. 2015. ChEC-seq kinetics discriminates transcription factor binding sites by DNA sequence and shape in vivo.</span><span style = "font-style:italic;">Nat Commun </span><span>6: 8733.</span></div></div>

[STEPS]
?. [Cell harvest]
Grow 50 ml of yeast cells in preferred conditions to OD600 = 0.5 – 0.7.
?. [Cell harvest]
When the culture approaches the desired OD600 set up a heat block or water bath to 30°C and prepare Buffer A.
?. [Cell harvest]
Harvest cells in a 50 ml tube (2000 x g, 3 min).
?. [Cell harvest]
Resuspend cells in 1 ml of Buffer A and transfer suspension to a 1.5 ml tube.
?. [Cell harvest]
Pellet cells (1500 x g, 30 sec) and resuspend in 1 ml of Buffer A.
?. [Cell harvest]
Repeat step 5 once. Pellet cells (1500 x g, 30 sec).
?. [DNA cleavage]
Resuspend cell pellet in 570 μl of Buffer A. Add 30 μl 2% digitonin (0.1% final), mix and permeabilize cells at 30°C for 5 min in a heat block with shaking (900 rpm).
?. [DNA cleavage]
Transfer 100 μl of cell suspension to an appropriate ‘stop tube’ (control sample) and mix.
We do not routinely sequence the control sample since it does not provide any advantage in data interpretation. In our experience in case of factors which show multiple genomic binding sites a certain enrichment of specific signals can be expected to be seen for the control sample but this minimal MNase activity is not affecting cells well-being in any noticeable way.
?. [DNA cleavage]
Add 4 μl of 25 mM CaCl2 (0.2 mM final concentration) to 500 μl of the remaining cell suspension and vortex briefly to mix (low to medium setting). Immediately return the tube to the heat block and start a timer. Keep shaking at 900 rpm.
0.2 mM final CaCl2 concentration is 10x lower than found in other protocols. We found that this approach limits the background digestion, at the same time allowing for a longer incubation, which in turn makes it feasible to process multiple samples at the same time.
?. [DNA cleavage]
After five minutes transfer 100 μl of cell suspension to an appropriate ‘stop tube’ and mix.
The remaining 400 μl of cell suspension can be used to collect additional time points or technical replicates although we do not do it routinely.
?. [DNA cleavage]
Add 4 μl of 20 mg/ml Proteinase K to each collected sample, mix and incubate at 55°C for 30 min.
?. [DNA purification]
Add 200 μl of phenol/chloroform/isoamyl alcohol, vortex for 10 sec and centrifuge at a maximum speed for 5 min.
?. [DNA purification]
Remove ~175 μl of an aqueous phase to a new tube. Add 20 mg of glycogen and 500 μl of 100% ethanol. Vortex vigorously to mix and precipitate on dry ice for 10 min.
Protocol can be stopped at this point. Store samples at -80°C.
?. [DNA purification]
Centrifuge at a maximum speed for 10 min at 4°C to pellet DNA.
?. [DNA purification]
Discard supernatants and wash pellets with 1 ml 100% ethanol (add ethanol to a tube and remove immediately with a pipette).
?. [DNA purification]
Spin briefly and remove the residual ethanol with a pipette taking care not to disturb the pellet. Air-dry pellets for 3 min at room temperature.
?. [DNA purification]
Resuspend DNA in 29 μl TE buffer, add 1 μl of 10 mg/ml RNase A and incubate at 37°C for 15 min.
DNA pellets at this step are sometimes hard to resuspend. We found that spinning the tubes for a few minutes at maximum speed followed by vortexing usually helps. Residual pellet may sometimes be still seen but it is not a reason for concern.
?. [DNA purification]
Add 60 μl of Mag-Bind beads (2x beads to DNA ratio) and pipet up and down 10x. Incubate the mixture for 10 min at room temperature.
?. [DNA purification]
Place tubes on a magnetic rack and allow beads to collect for 2 min.
?. [DNA purification]
Transfer supernatant (~90 μl) ) to a new tube containing 106 μl of 10 mM Tris (pH 8.0) and 4 μl of 5 M NaCl (100 mM final concentration). Discard the beads.
Supernatant is enriched in short DNA fragments. Longer DNA fragments are captured on the beads and are not useful for further processing. We routinely use Mag-Bind beads, not Ampure XP beads found in other protocols, since they have similar performance for a significantly lower price.
?. [DNA purification]
Add 200 μl of phenol/chloroform/isoamyl alcohol, vortex for 10 sec and centrifuge at a maximum speed for 5 min.
?. [DNA purification]
Remove ~175 μl of an aqueous phase to a new tube. Add 20 mg of glycogen and 500 μl of 100% ethanol. Vortex vigorously to mix and precipitate on dry ice for 10 min.
Protocol can be stopped at this point. Store samples at -80°C.
?. [DNA purification]
Centrifuge at a maximum speed for 10 min at 4°C to pellet DNA.
?. [DNA purification]
Discard supernatants and wash pellets with 1 ml 70% ethanol (add ethanol to a tube and remove immediately with a pipette).
?. [DNA purification]
Spin briefly and remove the residual ethanol with a pipette taking care not to disturb the pellet. Air-dry pellets for 3 min at room temperature.
?. [DNA purification]
Resuspend DNA in 25 μl 10 mM Tris (pH 8.0).
Samples can be quantified using a high-sensitivity kit but this is not informative because of possible contamination with large DNA fragments, which are not efficiently amplified during library preparation and are not a good substrate for sequencing.
?. [DNA sequencing and data analysis]
For library preparation we use half of the sample volume (~12 μl), no matter the concentration.
Any DNA library kit is suitable as long as it does not limit the recovery of very short (
?. [DNA sequencing and data analysis]
Sequence libraries in a paired-end and 25 bp read length mode.
We routinely use 25 bp read length which is optimal for yeast genome size. 2-3 million reads provide sufficient coverage for a ChEC sample which allows for multiplexing of many samples. In practice, we usually pool together 48 samples for a sequencing on a single lane of a HiSeq 2500 system.
?. [DNA sequencing and data analysis]
We align sequencing reads to the sacCer3 S. cerevisiae genome assembly using Bowtie2.
?. [DNA sequencing and data analysis]
Resulting SAM files are converted to tag directories with the HOMER (http://homer.ucsd.edu) ‘makeTagDirectory’ tool. Peaks are called using HOMER ‘findPeaks’ tool with optional arguments set to ‘-o auto -C 0 L 2 F 2’, with the free MNase data used as a control. These settings use a default false discovery rate (0.1%) and require peaks to be enriched 2-fold over the control and 2-fold over the local background. Resulting peak files are converted to BED files using ‘pos2bed.pl’ program. For each peak, the peak summit is calculated as a mid-range between peak borders Peaks are assigned to promoters if their peak summit location is in the range from −300 to +100 bp relative to the TSS. In rare cases, where more than one peak is assigned to the particular promoter, the one closer to the TSS is used.
?. [DNA sequencing and data analysis]
For quantitative analysis coverage at each base pair of the S. cerevisiae genome is calculated as the number of reads that mapped at that position divided by the number of all D. melanogaster (spike-in) reads mapped in the sample and multiplied by 10000 (arbitrarily chosen number).