CUTANA™ Stop Buffer
Powered by BiozCUTANA™ Stop Buffer is used in CUT&RUN to terminate pAG-MNase activity and prevent over-digestion of released DNA fragments. The Stop Buffer is added following pAG-MNase incubation, and halts enzyme activity by chelating free calcium ions.
Figure 1: CUT&RUN DNA fragment size distribution analysis
CUT&RUN was performed as described in Figure 4. Library DNA was analyzed by Agilent TapeStation®. This analysis confirmed that mononucleosomes were predominantly enriched in CUT&RUN (~300 bp peaks represent 150 bp nucleosomes + sequencing adapters).
Figure 2: CUT&RUN genome-wide heatmaps
CUT&RUN was performed as described in Figure 4. Heatmaps show two replicates (“Rep”) of IgG, H3K4me3, and H3K27me3 control antibodies in aligned rows ranked by intensity (top to bottom) relative to the H3K4me3 Rep 1 reaction and colored such that red indicates high localized enrichment and blue denotes background signal.
Figure 3: Representative gene browser tracks
CUT&RUN was performed as described in Figure 4. A representative 175 kb window at the TRMT2A gene is shown for two replicates (“Rep”) of IgG, H3K4me3, and H3K27me3 control antibodies. Tracks show the expected genomic distribution for each target. Images were generated using the Integrative Genomics Viewer (IGV, Broad Institute).
Figure 4: CUT&RUN methods
CUT&RUN was performed using the CUTANA™ ChIC/CUT&RUN Kit starting with 500k K562 cells with 0.5 µg of IgG (EpiCypher 13-0042), H3K4me3 (EpiCypher 13-0060), H3K27me3 (EpiCypher 13-0055), or 0.125 µg of CTCF (EpiCypher 13-2014) antibodies in duplicate. Library preparation was performed with 5 ng of DNA (or the total amount recovered if less than 5 ng) using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001/14-1002). Libraries were run on an Illumina NextSeq2000 with paired-end sequencing (2×50 bp). Sample sequencing depth was 5.5/18.8 million reads (IgG Rep 1/Rep 2), 14.2/17.0 million reads (H3K4me3 Rep 1/Rep 2), 24.7/18.1 million reads (H3K27me3 Rep 1/Rep 2), and 8.6/5.5 million reads (CTCF Rep 1/Rep 2). Data were aligned to the T2T-CHM13v2.0 genome using Bowtie2. Data were filtered to remove duplicates, multi-aligned reads, and ENCODE DAC Exclusion List regions.
Figure 5: CUT&RUN methods
CUT&RUN was performed using the CUTANA™ ChIC/CUT&RUN Kit starting with 500k K562 cells with 0.5 µg of IgG (EpiCypher 13-0042), H3K4me3 (EpiCypher 13-0060), H3K27me3 (EpiCypher 13-0055), or 0.125 µg of CTCF (EpiCypher 13-2014) antibodies in duplicate. Library preparation was performed with 5 ng of DNA (or the total amount recovered if less than 5 ng) using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001/14-1002). Libraries were run on an Illumina NextSeq2000 with paired-end sequencing (2×50 bp). Sample sequencing depth was 5.5/18.8 million reads (IgG Rep 1/Rep 2), 14.2/17.0 million reads (H3K4me3 Rep 1/Rep 2), 24.7/18.1 million reads (H3K27me3 Rep 1/Rep 2), and 8.6/5.5 million reads (CTCF Rep 1/Rep 2). Data were aligned to the T2T-CHM13v2.0 genome using Bowtie2. Data were filtered to remove duplicates, multi-aligned reads, and ENCODE DAC Exclusion List regions.
Storage
Stable for 12 months at 4°C from date of receipt.
Formulation
340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 µg/mL RNase A, 50 µg/mL glycogen.
Instructions for Use
CUTANA™ Stop Buffer includes sufficient buffer for 48 reactions using the CUTANA™ CUT&RUN protocol (www.epicypher.com/protocols).
| Item | Cat. No. |
|---|---|
| CUTANA™ ChIC/CUT&RUN Kit | 14-1048/14-1048-24 |
| CUTANA™ CUT&RUN Library Prep Kit | 14-1001/14-1002 |
| CUTANA™ pAG-MNase for ChIC/CUT&RUN | 15-1016/15-1116 |
| CUTANA™ Concanavalin A Conjugated Paramagnetic Beads | 21-1401/21-1411 |
| CUTANA™ Nuclei Extraction Buffer | 21-1026 |
| CUTANA™ E. coli Spike-in DNA | 18-1401 |
| Magnetic Separation Rack, 0.2 mL/1.5 mL Tubes | 10-0008/10-0012 |
| CUTANA™ CUT&RUN 8-strip 0.2 mL Tubes | 10-0009 |
