For each CUT&RUN sample, 5 × 106 cells were cultured for 48 hours with drug or vehicle control in growth media, 10% FBS in RPMI1640 (Thermo Fisher Scientific, catalog no. SH3006803). Each sample was represented by biological triplicates. Samples were processed using the low-salt protocol (26 (link)) and pAG-MNase (EpiCypher, SKU:15–1016). Antibodies used for CUT&RUN were: RB1 (Thermo Fisher Scientific, no. PA5–27215), RBL1 (Invitrogen, no. PA5–84228), RBL2 (Atlas Antibodies, no. HPA019703), and IgG (CST, no. 3900S). DNA libraries were prepared using MicroPlex Library Preparation Kit v2 (Diagenode, catalog no. C05010014) then sequenced on the Illumina HiSeq 2500, 50bp paired-end reads. Sequence reads were aligned to h38 using TopHat.v2 (27 (link)). Peaking calling was performed using MACS2. Differentially bound peaks were determined using DiffBind. Peak annotation was performed using ChIPseeker. Transcription factor similarity scoring was performed using GIGGLE (28 (link)). Peaks were visualized using IGV.
Pag mnase
Manufactured by EpiCypher
PAG-MNase is a laboratory tool used for chromatin analysis. It contains a Protein A-coated agarose bead that is conjugated with the micrococcal nuclease (MNase) enzyme. This product can be used to isolate and study chromatin fragments.
Automatically generated - may contain errors
Lab products found in correlation
Nebnext ultra 2 dna library prep kit for, by Illumina (4 mentions)
Nebnext ultra 2 dna library prep kit, by New England Biolabs (3 mentions)
Cutana dna purification kit, by EpiCypher (3 mentions)
Edta free tablet, by Roche (2 mentions)
Activated concanavalin a, by EpiCypher (2 mentions)
E coli spike in dna, by EpiCypher (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Microplex library preparation kit v2, by Diagenode (1 mentions)
Cutana cut run library prep kit, by EpiCypher (1 mentions)
Concanavalin a beads, by EpiCypher (1 mentions)
Halt protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Novaseq 6000 sp100 flowcell, by Illumina (1 mentions)
Concanavalin a coated magnetic beads, by Bangs Laboratories (1 mentions)
H3k9ac, by Diagenode (1 mentions)
Rabbit igg, by Merck Group (1 mentions)
Digitonin, by Promega (1 mentions)
Minelute column, by Qiagen (1 mentions)
Neb monarch dna cleanup kit, by New England Biolabs (1 mentions)
Ab8895, by Abcam (1 mentions)
11 protocols using pag mnase
1
CUT&RUN Analysis of Rb Family in Cell Lines
2
Chromatin Immunoprecipitation of HNF1α in Pseudoislet Cells
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CUT&RUN was performed on 500,000 dispersed HNF1α-FLAG pseudoislet cells per condition using CUTANA ChIC/CUT&RUN protocol v3.1. Nuclei were extracted with nuclear extraction buffer (20 mM HEPES-KOH [pH 7.9]; 10 mM KCl; 0.1% Triton X-100; 20% glycerol; 1 mM MnCl2; 0.5 mM spermidine; 1× Halt protease inhibitor; Thermo Fisher Scientific) for 10 minutes on ice and immobilized onto Concanavalin-A beads (EpiCypher). After blocking and washes, samples were incubated with 0.5 μg of rabbit anti-FLAG (MilliporeSigma F7425) or rabbit anti-IgG (EpiCypher 13-0042) antibodies (Supplemental Table 2 ) overnight at 4°C. pAG-MNase (EpiCypher) was added to nuclei (1:20) and incubated at room temperature for 10 minutes. Targeted chromatin digestion was induced by adding 100 mM CaCl2 and nutating for 2 hours at 4°C. DNA fragments were purified using the CUTANA ChIC/CUT&RUN kit, according to the manufacturer’s instructions. DNA was resuspended in 0.1× Tris-EDTA buffer solution and used for library preparation with the CUTANA CUT&RUN Library Prep Kit (EpiCypher, 14-1001), according to the v1 manual. Libraries were sequenced as PE150 reads on the NovaSeq platform.
3
Cut&Run: Profiling Chromatin-Associated Proteins
4
CUT&RUN for Histone Modifications and Protein Targets
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MM.1S cells expressing either FLAG-3xHA or FLAG-3xHA-ADA2B were subjected to light crosslinking with 0.1% formaldehyde for 1 minute, followed by quenching with 125 mM glycine. Remaining steps of CUT&RUN were performed by the Epigenomics Profiling Core at MD Anderson Cancer Center according to published protocols (Meers et al. 2019 (link)) with some modifications. Briefly, 500,000 cells were immobilized to activated Concanavalin A-coated magnetic beads (Bangs Laboratories) followed by permeabilization with wash buffer containing Digitonin (Promega). Samples were incubated with rabbit IgG (Millipore), H3K9ac (Diagenode) and HA (Cell Signaling Technology) antibodies overnight at 4°C. Targeted chromatin digestion was achieved by pAG-MNase (EpiCypher) binding for 10 minutes at room temperature followed by incubation with CaCl2 at 4°C. DNA fragments were purified using MinElute columns (Qiagen). Libraries were prepared using NEBNext Ultra II DNA Library prep kit (New England Biolabs) following manufacturer’s instructions for H3K9ac CUT&RUN DNA, and a modified library preparation protocol for HA CUT&RUN DNA as described previously (Liu et al. 2018 (link)). Libraries were sequenced at the Advanced Technology Genomics Core at MD Anderson Cancer Center using Illumina NovaSeq 6000 SP-100 flow cell to obtain 50bp paired-end reads.
5
Cut&Run: Profiling Chromatin-Associated Proteins
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Cut and Run was performed as described in EpiCypher® CUTANA® CUT&RUN protocol with minor modifications. Briefly, 0.5 million live HCT116 or HeLa cells were harvested and resuspended in 100ul wash buffer [20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, supplemented with Protease Inhibitor EDTA-Free tablet (Roche 11836170001)]. Activated Concanavalin A (EpiCypher 21–1401) was incubated with cells at room temperature for 10min to let the cells bind to the beads. For each target protein factor, 0.5μg antibody was added to each sample and incubated in the antibody buffer (wash buffer +0.01% Digitonin and 2 mM EDTA) at 4°C for 4–6hr. The beads were then washed twice with digitonin buffer [wash buffer +0.01% Digitonin], and 2.5 μL pAG-MNase (EpiCypher, 15–1116) was added to each sample. After ten minutes of incubation at room temperature, excessive pAG-MNase was washed out by a two-time digitonin buffer wash. Then targeted chromatin was digested and released from cells by 2 h of incubation with the presence of 2 mM CaCl2 at 4°C, which were collected from the supernatant, were subjected to phenol/chloroform DNA extraction, and finally to sequencing library construction using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (NEB, E7645L).
6
CUT&RUN for Histone Modifications
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OCI-Ly7 cells were subjected to CUT&RUN assay according to the EpiCypher CUTANA CUT&RUN Protocol79 . In brief, 500 K cells were immobilized onto activated ConA magnetic beads (Bangs Laboratories, BP531), followed by permeabilization and incubation with 0.5 ug of antibodies (H3K4me1, 13-0040; H3K4me3, 13-0028; or H3K27ac, 13-0045; SNAP-Certified for CUT&RUN and ChIP by EpiCypher) overnight at 4 °C. On the next day, the cell-bead slurry was washed and incubated with pAG-MNase (1:20 dilution, EpiCypher, 15-1116) for 10 min at RT. MNase was then activated by addition of CaCl2 to cleave targeted chromatin for 2 h at 4 °C. After chromatin digestion, MNase activity was stopped and chromatin fragments released into supernatant were purified using the NEB Monarch DNA Cleanup Kit (NEB, T1030) per manufacturer’s instruction. 10 ng DNA was subjected to library preparation using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645) according to the manufacturer’s protocol. Libraries were loaded onto the Illumina HiSeq for pair-end 150 bp sequencing with a sequencing depth of around 6 M reads per sample.
7
CUT&RUN Analysis of Chromatin Marks
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CUT and release using nuclease (CUT&RUN) was performed as previously described98 (link)–100 (link) with slight modifications. In brief, 500,000 cells per condition were bound to activated ConA beads (EpiCypher 21-1401). Next, the ConA bead–cell mixture was resuspended in a cold antibody buffer and FLI-1-(ab133485; 1 μg per sample) antibody or 0.5 μg H3K4me3 (EpiCyper, 13-0041) as positive and 0.5 μg IgG (EpiCypher, 13-0042) as negative control were per sample added overnight. pAG-MNase (EpiCypher, 15-1016) was then added to each reaction to allow binding to the antibody-labelled chromatin. E. coli spike-in DNA (EpiCypher, 18-1401) was added following MNase activation. Subsequently, targeted chromatin was digested and released by the addition of CaCl2. The fragmented chromatin was purified using a CUTANA DNA Purification kit (EpiCypher, 14-0050). Quantification, library preparation and sequencing were performed by the genomics core at Dana-Farber Cancer Institute.
8
CUT&RUN Profiling of Histone Modifications
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CUT&RUN assays were performed as previously described in uninduced iGMPs48 (link)–50 . In summary, 0.5 million cells were collected for each sample. Cells were washed twice (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, protease inhibitor) and bound to concavalin A beads (EpiCypher 21-1401). Cells were then permeabilized and incubated overnight at 4 °C with primary antibody at a 1:100 dilution (wash buffer + 0.01% digitonin, 2 mM EDTA). The following antibodies were used H3K4me1 (ab8895, Abcam), H3K27me3 (9733S, Cell Signaling), H3K9me3 (ab8898, Abcam), and IgG (2792S, Cell Signaling). Cells were then washed twice with 0.01% digitonin in wash buffer. pAG-MNase (15–1116, EpiCypher) was then used at 1:20 dilution for each 50 μL CUT&RUN reaction and incubated for 10 min at room temperature. Cells were then washed twice with digitonin buffer and 2 mM CaCl2 was added for 2 h at 4 °C. Stop buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 µg/mL RNase A, 50 µg/mL glycogen) was added at 1.5× dilution and incubated for 10 min at 37 °C. DNA was purified with CUTANA DNA purification kit (EpiCypher, 14-0050). Library construction was performed using the NEBNext UltraII DNA Library Prep Kit from NEB (E7645S). Indexed samples were run using the Illumina HiSeq 4000 platform with paired-end sequencing.
9
dTAG-47 Chromatin Immunoprecipitation
10
CUT&RUN Profiling of Transcription Factors
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CUT&RUN assay was performed using the EpiCypher CUTANA™ ChIC/CUT&RUN kit v2.1 according to manufacturer’s protocol. In brief, 106 cells were harvested for each condition, washed with 1X PBS, and resuspended in wash buffer, followed by incubation at RT for 10 min with activated Concanavalin A (ConA) beads. Cells were then permeabilized and incubated overnight on a nutator at 4°C with 1 μg of normal rabbit IgG (EpiCypher), KLF4 (proteintech, 1180–1-AP), or GRIP1 (Novus Biologicals, NB100–1756) antibody. Cell-bead antibody complex was then washed with cell permeabilization buffer and incubated with pAG-MNase (EpiCypher), followed by addition of 2 mM CaCl2 and incubation for 2 h at 4°C. Stop buffer was added to the reaction mixture and DNA fragments were released and purified with a CUTANA DNA purification kit (EpiCypher). DNA library construction was performed using the NEBNext Ultra II DNA Library Prep Kit from NEB (E7645L). The libraries were sequenced on Illumina NovaSeq 6000 system (50 bp paired-end) at the Genomics Resources Core Facility at Weill Cornell Medicine.
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