Drosophila Spike-In Control Kit for CUT&Tag (Rabbit) Protocol

! This signifies an important step in the protocol regarding volume changes based on the number of CUT&Tag reactions being performed.

NOTE: Before starting this protocol, activate Concanavalin A Beads and prepare cell or tissue samples by following Sections I and II in our CUT&Tag Assay Kit #77552 protocol. Then proceed to Section I.

I. Addition of Drosophila Spike-In Nuclei Control for Sample Normalization

Before Starting:

• Thaw the Drosophila Spike-In Nuclei Control at room temperature. Each vial provides sufficient spike-in nuclei for up to 8 reactions, based on the recommended volume of 20 µL of spike-in nuclei per reaction containing 100,000-250,000 cells or 1-2.5 mg of tissue.
• If more than one vial of Drosophila Spike-In Nuclei Control is needed within an experiment, pool the thawed vials together to ensure consistency in the number of spike-in nuclei added per reaction.
• Minimize freezing and thawing of the Drosophila Spike-In Nuclei Control. Aliquot into smaller volumes to limit freeze-thaw cycles to four or fewer. Exceeding this may reduce the mapping rate to the Drosophila genome and require higher volumes of spike-in nuclei per reaction; for example, nine freeze-thaw cycles can reduce the Drosophila mapping rate by ~50%.

1. Aliquot 100 µL of cell suspension, as prepared in Section II or Appendices in CUT&Tag Assay Kit #77552 protocol, into separate 1.5 mL tubes for each reaction.
2. Add 20 µL of Drosophila Spike-In Nuclei Control to each reaction containing 100,000-250,000 cells or 1-2.5 mg of tissue. This ratio of spike-in nuclei to cells typically results in Drosophila spike-in normalization reads representing ~0.5-10% of the total sequencing reads for highly abundant targets.

   NOTE: The optimal volume of Drosophila Spike-In Nuclei Control for CUT&Tag varies and should be optimized by the user so that the spike-in normalization reads represent ~0.5-10% of the total sequencing reads. If using less than 100,000 cells or 1 mg of tissue, proportionally reduce the volume of Drosophila Spike-In Nuclei Control, maintaining a ratio of 20 µL per 100,000 cells or per 1 mg tissue (e.g., 10 µL of Drosophila Spike-In Nuclei for 50,000 cells or 1 µL of Drosophila Spike-In Nuclei for 5,000 cells). Additionally, if the target of interest is low-abundance (e.g., certain transcription factors or cofactors), the volume of spike-in nuclei may need to be reduced by 2-10-fold. For example, use 2-10 µL of Drosophila Spike-In Nuclei Control per reaction containing 100,000-250,000 cells or 1-2.5 mg of tissue.

3. Mix gently by pipetting up and down.
4. Immediately proceed to Section II.

II. Binding of Concanavalin A Beads and Primary Antibody

NOTE: For all incubation steps in this section, it is not necessary to mix samples by rocking or rotation. Instead, we recommend simply placing sample tubes in a rack at the designated temperatures. Mixing the samples during the incubation steps does not increase the performance of the assay. Instead, rotation or rocking the samples may lead to increased bead clumping and bead loss due to potential sticking on the tube walls and caps.

Before Starting:

! All buffer volumes should be increased proportionally based on the number of CUT&Tag reactions being performed.

• Warm Digitonin Solution #16359 at 90-100°C for 5 min, and make sure it is completely thawed and in solution. Immediately place the thawed Digitonin Solution #16359 on ice during use. Store at -20°C when finished for the day.
• Thoroughly thaw Protease Inhibitor Cocktail (200X) #7012 and 100X Spermidine #27287 before use and store them at -20°C when finished for the day. Please note that the Protease Inhibitor Cocktail (200X) #7012 will refreeze when placed on ice due to the presence of DMSO.
• Prepare 100 µL of Complete Antibody Binding Buffer per reaction and place it on ice.

1. Equilibrate the activated Concanavalin A Beads prepared in Section I Step 7 in the CUT&Tag Assay Kit #77552 protocol to room temperature and mix thoroughly by gently pipetting up and down.
2. Add 10 µL bead suspension to each tube of cells + Drosophila Spike-In Nuclei suspension prepared in Section I Step 3.
3. Mix the samples well by pipetting up and down. Incubate the sample for 5 min at room temperature.
4. Place tubes on the magnetic rack for 30 sec-2 min, then remove and discard the supernatant.
5. Remove tubes from the magnetic rack. Add 100 µL of Complete Antibody Binding Buffer per reaction, gently mix by pipetting up and down, and place on ice.
6. Add the appropriate amount of test antibody and 1 µL of H2Av Rabbit Monoclonal Antibody to each reaction tube and mix gently by pipetting up and down.

   NOTE: The amount of antibody required for CUT&Tag varies and should be determined by the user. For the positive control Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit Monoclonal Antibody #9751, or the negative control Normal Rabbit IgG #2729 or Normal Mouse IgG #68860, add 2 µL of antibody to the reaction. If possible, we highly recommend using CUT&Tag-validated antibodies in the assay. CST offers a selection of CUT&Tag-validated antibodies with supporting data and appropriate dilution ratios.

7. Incubate samples at room temperature for 1 hr. This step can be extended to overnight at 4°C.
8. Continue with the CUT&Tag experiment by following Sections IV-VII in the CUT&Tag Assay Kit #77552.

III. Next-Generation Sequencing (NGS) Analysis for Sample Normalization

For sample normalization, map CUT&Tag sequencing data from all samples to both the test genome (e.g., human, mouse, or other) and the Drosophila melanogaster genome. Calculate the ratio of unique spike-in reads to total uniquely aligned reads for each sample. Select the sample with the lowest ratio (e.g., Sample 4 in the table below) as the Reference Sample and calculate normalization factors for the other samples using the equation provided. Apply these factors during bioinformatics analysis by either scaling bigWig files (e.g., with deepTools bamCoverage) or downsampling the number of unique reads aligned to the test genome for each sample. If sequencing depth is consistent across samples, the normalization factor can also be calculated by using the absolute number of spike-in reads instead of the ratio of spike-in reads to total reads.

An Example of Sample Normalization for NGS Assay using CTCF (D1A7) Rabbit Monoclonal Antibody #3417 In A Cell Titration Experiment

Normalization Factor for NGS = Ratio of unique spike-in reads to total uniquely aligned reads from Reference Sample / Ratio of unique spike-in reads to total uniquely aligned reads from the Other Sample

APPENDIX: Troubleshooting Guide

For Research Use Only. Not for Use in Diagnostic Procedures.

Please contact us for additional technical support.