1. Chromosomes and Gene Expression
  2. Genetics and Genomics
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Improved CUT&RUN chromatin profiling tools

  1. Michael P Meers
  2. Terri D Bryson
  3. Jorja G Henikoff
  4. Steven Henikoff  Is a corresponding author
  1. Fred Hutchinson Cancer Research Center, United States
  2. Howard Hughes Medical Institute, United States
Research Advance
Cite this article as: eLife 2019;8:e46314 doi: 10.7554/eLife.46314
5 figures, 2 tables and 1 additional file

Figures

Figure 1 with 2 supplements
An improved fusion protein for CUT&RUN.

(A) Schematic diagram (not to scale) showing improvements to the pA-MNase fusion protein, which include addition of the C2 Protein G IgG binding domain, a 6-histidine tag for purification and a hemagglutinin tag (HA) for immunoprecipitation. (B) The Protein A/G hybrid fusion results in high-efficiency CUT&RUN for both rabbit and mouse primary antibodies. CUT&RUN for both rabbit and mouse RNAPII-Ser5phosphate using pAG/MNase were extracted from either the supernatant or the total cellular extract. Tracks are shown for the histone gene cluster at Chr6:26,000,000–26,300,000, where NPAT is a transcription factor that co-activates histone genes. Tracks for 2’ and 10’ time points are displayed at the same scale for each antibody and for both supernatant (supn) or total DNA extraction protocols.

https://doi.org/10.7554/eLife.46314.002
Figure 1—figure supplement 1
An improved fusion protein for CUT&RUN.

(A) Plasmid map of pAG-ERH-MNase-6xHIS-HA. (B) Coomassie-stained gel of fusion protein eluted from nickel-agarose.

https://doi.org/10.7554/eLife.46314.003
Figure 1—figure supplement 2
pAG/MNase titration.

(A) K562 cells were incubated with an antibody to H3K27me3 (CST #9733 Rabbit monoclonal), washed twice with 1 ml Dig-wash. The sample was split into aliquots for incubation with pA/MNase at the recommended concentration and a serial dilution of pAG/MNase, followed by 3 1 ml washes. After 30 min using the standard protocol, lImit digestions are seen at all dilutions for this abundant epitope, indicating that the amount of fusion protein used in this experiment was in excess. (B) Representative tracks from these samples on the same normalized count scale show consistently low CUT&RUN backgrounds with excess pAG/MNase, which indicates that washes are sufficient to minimize non-specific background cleavages. ENCODE ChIP-seq tracks are shown for comparison, where USC used CST #9733, and Broad Institute used Millipore 07–449.

https://doi.org/10.7554/eLife.46314.004
Targeted fragments are not released during digestion using high-calcium/low-salt conditions.

CUT&RUN was performed using either the high-Ca++/low-salt (Ca++) or the standard (Std) method with antibodies to three different epitopes. DNA was extracted from supernatants, where no elution was carried out for the Ca++ samples. Although high yields of nucleosomal ladder DNA eluted from the supernatants using the standard method, no DNA was detectable in the supernatant using the high-Ca++/low salt method when the elution step was omitted. Left, Tapestation images from indicated lanes; Right, Densitometry of the same lanes.

https://doi.org/10.7554/eLife.46314.005
Similar performance using pA/MNase and pAG/MNase.

(A) CUT&RUN was performed with an antibody to H3K27ac (Millipore MABE647) and to CTCF (Millipore 07–729) with digestion over a 1 to 27 min range as indicated using pAG/MNase with the high-Ca++/low-salt protocol. Correlation matrix comparing peak overlaps for time points and fusion constructs. The datasets were pooled and MACS2 was used with default parameters to call peaks, excluding those in repeat-masked intervals and those where peaks overlapped with the top 1% of IgG occupancies, for a total of 52,425 peaks. Peak positions were scored for each dataset and correlations (R2 values shown along the diagonal and displayed with Java TreeView v.1.16r2, contrast = 1.25) were calculated between peak vectors. IgG and H3K27me3 (me3) negative controls were similarly scored. (B) Same as A, except the antibody was to CTCF. A set of 9403 sites with a CTCF motif within a hypersensitive site was used (Skene and Henikoff, 2017). High correlations between all time points demonstrate the uniformity of digestion over a 27-fold range. (C) Representative tracks from datasets used for panels A and B showing a 100 kb region that includes a histone locus cluster (chr6:25,972,600–26,072,600).

https://doi.org/10.7554/eLife.46314.006
Figure 4 with 2 supplements
Consistent peak definition with high-Ca++/low salt digestion.

(A) H3K27ac CUT&RUN time-course experiments were performed with an Abcam 4729 rabbit polyclonal antibody, following either the standard protocol or the low-salt/high-calcium (High-Ca++) protocol. Samples of 5 million fragments from the 10 H3K27ac datasets were pooled and MACS2 called 36,529 peaks. Peak positions were scored for each dataset and correlations (R2 values shown along the diagonal) were calculated between peak vectors. IgG and H3K27me3 (me3) negative controls were similarly scored. Higher correlations between the High-Ca++ than the Standard time points indicates improved uniformity of digestion over the ~100 fold range of digestion times. (B) Tracks from a representative 200 kb region around the HoxB locus. (C) Fraction of reads in peaks (Frip) plots for each time point after down-sampling (5 million, 2.5 million, 1.25 million, 625,000 and 312,500), showing consistently higher Frip values for Ca++ (red) than Std (blue).

https://doi.org/10.7554/eLife.46314.007
Figure 4—figure supplement 1
CUT&RUN consistency with high-Ca++/low salt digestion and total DNA extraction.

(A) H3K4me2 CUT&RUN time points with digestions using either the standard protocol or the high-calcium/low-salt protocol with either supernatant or total DNA extraction. To construct the correlation matrix, all 8 H3K4me2 datasets were pooled and MACS2 was used to call peaks, which yielded 64,156 peaks. Peak positions were scored for each dataset and correlations (R2 displayed with Java TreeView v.1.16r2, contrast = 1.25) were calculated between peak vectors. IgG and H3K27me3 (me3) negative controls were similarly scored. (B) Same as Figure 4B. (C) Same as Figure 4C.

https://doi.org/10.7554/eLife.46314.008
Figure 4—figure supplement 2
Tapestation analyses of an H3K27ac digestion time-course series.

CUT&RUN with the low-salt/high-calcium protocol results in fragment release within 20 s at 0°C.

https://doi.org/10.7554/eLife.46314.009
E. coli carry-over DNA of pA/MNase and pAG/MNase can substitute for spike-in calibration.

(A) Fragments from a CUT&RUN K562 cell experiment (GSE104550 20170426) using antibodies against H3K27me3 (100–8,000 cells) and CTCF (1,000–100,000 cells) were mapped to the repeat-masked genome of S. cerevisae and the full genome of E. coli. Standard digestion was followed by supernatant release and extraction. (B) Same as A using antibodies against multiple epitopes of varying abundances, with high-calcium/low-salt digestion followed by supernatant release and extraction. (C) Same as B except using standard digestion conditions and total DNA extraction. The S. cerevisiae spike-in DNA was left out for one sample (blue square). From top to bottom, antibodies are: NPAT Thermo PA5-66839, Myc: CST Rabbit Mab #13987, CTD: PolII CTD Abcam 8WG16, RNAPII-Ser5: Abcam 5408 (mouse), RNAPII-Ser2: CST E1Z3G, CTCF Millipore 07–729, RNAPII-Ser5: CST D9N5I (rabbit), H3K4me2: Upstate 07–030. (D) Same as C except using high-calcium/low-salt digestion and total DNA extraction. From top to bottom, antibodies are: CTCF Millipore 07–729, NPAT Thermo PA5-66839, Myc: CST Rabbit Mab #13987, CTD: PolII CTD Abcam 8WG16, RNAPII-Ser5: Abcam 5408 (mouse), RNAPII-Ser5: CST D9N5I (rabbit), RNAPII-Ser2: CST E1Z3G, H3K4me2: Upstate 07–030.

https://doi.org/10.7554/eLife.46314.010

Tables

Table 1
Carry-over E. coli DNA correlates closely with the heterologous spike-in for both fusion proteins and both low-salt/high-calcium and standard digestion conditions.

CUT&RUN was performed for H3K27me3 in parallel for pA/MNase Batch #6 (pA), pAG/MNase (pAG) using both low-salt/high-calcium (lo-hi) and standard (std) CUT&RUN digestion conditions. Each sample started with ~700,000 cells and 10 µL of bead slurry. Also varied in this experiment was addition of antibody followed by bead addition (Ab first) and addition of 0.1% BSA in the antibody buffer (BSA). Adding antibody first led to increased recovery of both yeast and E. coli DNA relative to human DNA, indicative of loss of cells prior to addition of fusion protein, possibly caused by loss of digitonin solubilization of membrane sugars.

https://doi.org/10.7554/eLife.46314.011
H3K27me3Ab firstBSAHumanYeastE. coliCorr (Sc:Ec)
pA lo-hi591398374334550.92
pA lo-hi+77480038584988
pA lo-hi+5202278228816110
pA lo-hi++5178086180418759
pA std601334759524620.99
pA std+60050808592295
pA std+4104736262421236
pA std++3972820232819245
pAG lo-hi69998027894040.94
pAG lo-hi+6374939642467
pAG lo-hi+414040715651291
pAG lo-hi++405869323825289
pAG std75141273085670.90
pAG std+5935592355125
pAG std+45941531271555
pAG std++537961025091353
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (Human)K562ATCC#CCL-243RRID: CVCL_0004
Biological sample (Escherichia coli)JM101 cellsAgilent#200234
Antibodyrabbit polyclonal anti-NPATThermoPA5-66839Concentration: 1:100; RRID:AB_2663287
Antibodyguinea pig polyclonal anti-rabbit IgGAntibodies OnlineABIN101961Concentration: 1:100; RRID: AB_10775589
Antibodyrabbit polyclonal anti-mouse IgGAbcam46540Concentration: 1:100; RRID: AB_2614925
Antibodyrabbit monoclonal anti-RNAPII-Ser5Cell SignalingD9N51Concentration: 1:100
Antibodymouse monoclonal anti-RNAPII-Ser5Abcam5408Concentration: 1:100; RRID:AB_304868
Antibodyrabbit monoclonal anti-H3K27me3Cell Signaling9733Concentration: 1:100; RRID: AB_2616029
Antibodyrabbit polyclonal anti-H3K4me2Upstate07–730Concentration: 1:100; RRID: AB_11213050
Antibodyrabbit monoclonal anti-H3K27acMilliporeMABE647Concentration: 1:100;
Antibodyrabbit polyclonal anti-H3K27acAbcam4729Concentration: 1:100; RRID: AB_2118291
Antibodyrabbit polyclonal anti-CTCFMillipore07–729Concentration: 1:100; RRID: AB_441965
Recombinant DNA reagentAG-ERH-MNase-6xHIS-HA (plasmid)Progenitors: pK19-pA-MN; gBlocks
Recombinant DNA reagentpK19-pA-MNSchmid et al., 2004Gift from author
Sequence-based reagentgBlock Hemagglutinin and 6-histidine tags; gattacaGAAGACAACGCTGATTCAGGTCAAGGCGGtGGTGGcTCTGGgGGcGGgGGcTCGGGtGGtGGgGGcTCAcaccatcaccatcaccatGGCGGtGGTGGcTCTTACCCATACGATGTTCCAGATTACGCTtaatgaGGATCCgattacaIntegrated DNA Technologies (IDT)
Sequence-based reagentgBLOCK PrtG_ERH Codon optimized; AGCAGAAGCTAAAAAGCTAAACGATGCTCAAGCACCAAAAACAACTTATAAATTAGTCATCAACGGGAAAACGCTGAAGGGTGAAACCACGACAGAGGCCGTAGATGCGGAGACAGCGGAGCGCCACTTTAAGCAATACGCGAATGATAACGGTGTAGACGGCGAGTGGACCTACGACGACGCGACAAAGACCTTTACCGTCACGGAGAAACCTGAGGTTATCGACGCGTCTGAGTTGACGC
CAGCCGTAGATGACGATAAAGAATTCGCAACTTCAACTAAAAAATTAC
Integrated DNA Technologies (IDT)
Peptide, recombinant proteinpA/MNaseSchmid et al., 2004purified as described inSchmid et al., 2004 and supplementary
Peptide, recombinant proteinpAG/MNaseThis paperPurified from modified plasmid pAG-ERH-MNase-6xHIS-HA in
S Henikoff Lab
Commercial assay or kitPull-Down PolyHis Protein:Protein Interaction KitThermo#21277
OtherConcanavalin A coated magnetic beadsBangs Laboratories#BP-531
OtherGibson AssemblyNew England Biolabs#E2611
OtherChicken egg white lysozymeEMD Millipore#71412
OtherZwittergent 3–10 detergent (0.03%)EMD Millipore#693021
Chemical compound, drugDigitoninEMD Millipore#300410
Chemical compound, drugRoche Complete Protease Inhibitor EDTA-free tabletsSigma Aldrich5056489001
Chemical compound, drugRNase A Dnase- and protease-freeThermoENO53110 mg/ml
Chemical compound, drugProteinase KThermoEO0492
Chemical compound, drugGlycogenSigma-Aldrich10930193001
Chemical compound, drugSpermidineSigma-Aldrich#S0266

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