CUT&RUN was performed using the CUTANA CUT&RUN Protocol (www.epicypher.com ) which is an optimized version of that previously described55 (link),56 (link). For each sample, 5 × 105 cells were immobilized onto Concanavalin-A beads (EpiCypher catalog no. 21-1401) and incubated overnight (4 °C with gentle rocking) with 0.5 µg of antibody. CUT&RUN-enriched DNA was purified and 10 ng used to prepare sequencing libraries with the KAPA HTP/LTP Library Preparation Kits (Roche catalog no. 07961880001). Libraries were sequenced with Illumina HiSeq 4000 or NovaSeq 6000 system according to the manufacturer’s instructions. Paired end fastq files were aligned to the hg38 or mm10 reference genome using the Bowtie v.2 algorithm. Only uniquely aligned reads were retained for subsequent analyses.
Concanavalin a beads
Manufactured by EpiCypher
Concanavalin-A beads are affinity chromatography beads. They are used for the purification of glycoproteins, lectins, and other carbohydrate-binding biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Cutana cut run library prep kit, by EpiCypher (2 mentions)
Hiseq 4000, by Illumina (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Pag mnase, by EpiCypher (1 mentions)
Halt protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Monoclonal anti flag m2, by Merck Group (1 mentions)
Cutana pag mnase, by EpiCypher (1 mentions)
Cutana chic cut run kit, by EpiCypher (1 mentions)
Dna clean concentrator 5, by Zymo Research (1 mentions)
Pag micronuclease, by EpiCypher (1 mentions)
5 protocols using concanavalin a beads
1
CUT&RUN: Chromatin Profiling Protocol
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 Protocol for K562 Cell Nuclei
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CUT&RUN was performed on 500,000 native nuclei extracted from K562 cells using CUTANA protocol v1.5.1 (http://www.epicypher.com ), which is an optimized version of that previously described (Skene et al. 2018 (link)). For each sample, nuclei were extracted by incubating cells on ice for 10 min in nuclei extraction buffer (NE: 20 mM HEPES–KOH at pH 7.9; 10 mM KCl; 0.1% Triton X-100; 20% glycerol; 0.5 mM spermidine; 1× complete protease inhibitor; Roche 11836170001), collecting by centrifugation (600g, 3 min, 4°C), discarding the supernatant, and resuspending at [100 µL/500 K nuclei] sample in NE buffer. For each target, 500,000 nuclei were immobilized onto Concanavalin-A beads (EpiCypher #21-1401) and incubated overnight (4°C with gentle rocking) with 1 µg of antibody (for all 40 PCRP antibodies as above; RbIgG [EpiCypher 13-0042, lot 20036001-52]; MsIgG [Invitrogen 10400C, lot VD293456]; CTCF [Millipore 07-729, lot 3205452]).
4
CUT&RUN of FLAG-tagged CIART in hPSC-CMs
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CUT&RUN was performed using CUTANA ChIC/CUT&RUN Kit (Epicypher) according to the user manual. Briefly, differentiation day 30 hPSC-CMs were transduced with lentiviruses encoding FLAG-tagged cDNA to the open reading frame of human CIART. Seven days after transduction, 1 × 106 cells per sample were washed and bound to 11 μl of activated Concanavalin A beads (Epicypher). The bead-bound cells were incubated with monoclonal anti-FLAG M2 (Sigma Aldrich; 1:100) at 4 °C overnight. After washing, the cells were incubated with CUTANA pAG-MNase (Epicypher) for 10 min, targeted chromatin tagmentation was initiated by the addition of 100 mM CaCl2 and allowed to proceed for 2 h at 4 °C, and then stop buffer containing 0.5 ng Escherichia coli spike-in DNA was added to each sample. Released chromatin fragments were purified using DNA Clean & Concentrator-5 (Zymo Research). Libraries were generated using a NEBNext ultra II DNA library prep kit and sequenced on an Illumina NovaSeq (PE-00) at the Weill Cornell Medical College Genomics Core.
5
Automated CUT&RUN for Histone Modifications
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CUTANA CUT&RUN was performed with RPE-1 and DLD-1 samples on an automated protocol (autoCUT&RUN) derived from those previously described45 (link),46 (link). In brief, for each CUT&RUN reaction, 500,000 cells (5 million cells per ml prepared in 20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1 mM trichostatin A, 1× EDTA/EGTA-free complete protease inhibitor) were dispensed to individual wells of a 96-well plate, immobilized onto concanavalin-A beads (EpiCypher) and incubated overnight (4 °C) with 0.5 µg antibody (IgG, H3K4me3, H3K27me3, H3K27ac) (all antibodies validated to histone PTM-defined SNAP-ChIP nucleosome standards). pAG-micronuclease (EpiCypher) was added and activated (2 h at 4 °C), and CUT&RUN-enriched DNA was purified using Serapure beads after mixing at a 2:1 (bead:DNA) ratio. Recovered DNA was quantified using PicoGreen and reactions were normalized to 5 ng DNA (or the entirety of the reaction if less than 5 ng DNA was recovered) before preparing sequencing libraries (CUTANA CUT&RUN Library Prep kit; EpiCypher). All CUT&RUN steps were optimized and performed on Tecan Freedom EVO robotics platforms with gentle rocking for incubation steps and magnetic capture for medium exchange and washing steps as described previously45 (link),46 (link).
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