CUT&Tag-IT™* Service
Sample Preparation
Active Motif recommends preparing 400,000 cells for each CUT&Tag reaction. If cells arelimited, less may be acceptable with prior approval. If examining multiple targets withCUT&Tag, please provide separate tubes of cells for each reaction and approximately
the same number of cells per tube within a project.
NOTE: Cells must be cryopreserved. Flash frozen cell pellets are not compatible with thisservice. For high quality data, we recommend sending samples with >70% cell viability.
Please thaw a test sample to test post-thaw viability.

Reagents
Enzyme Free Cell Dissociation Solution Hank′s Based (1X)- (Millipore-Sigma, Cat. No.
S-004-M) or equivalent.

Cryopreservation of cells
1. Incubate Mr. Frosty or equivalent device at 4oC for a minimum of 1-hour prior to use.
2. For healthy adherent cells lines, use Enzyme-Free Cell Dissociation Solution and scrape
cells with a rubber policeman or by pipetting. DO NOT use enzyme-based dissociation
methods. For healthy suspension cells, transfer cells in growth media to a conical tube forpelleting.
3. Harvest cells at room temperature and count cells using hemocytometer or equivalentmethod to determine volume needed to achieve the proper concentration of cells forcryopreservation. While quantifying, keep cells on ice. Using low-bind microcentrifuge
tubes may help avoid potential sample loss.
4. Centrifuge at 500 x g at 4°C to pellet the cells and remove supernatant.
5. Resuspend cells in 500 μL of ice-cold cryopreservation solution – 50% FBS/40% growth
media/10% DMSO. Transfer 500 μL to a 1.5 mL Eppendorf tube on ice.
6. Freeze the cells by transferring the tubes to a pre-chilled Mr. Frosty container or
equivalent device, like the one depicted below and place at -80o C.

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Europe +32 (0)2 653 0001
www.activemotif.com
CUT&Tag-IT™ Service Sample Preparation Protocol

Japan +81 (0)3 5225 3638
Rev G091124

7. If necessary, an alternate approach is to place the tubes upright in a styrofoam container.
Close the styrofoam container with the styrofoam top and then place at -80o C.

Tissue preparation protocols
If you are submitting tissues for CUT&Tag, freeze the tissue according to one of theprotocols below. We ask for 10-30 mg frozen tissue per reaction.
Liquid Nitrogen
1. Excise the tissue from the animal and place in a microfuge tube.
2. Submerge in liquid nitrogen for 2 minutes.
3. Store at -80oC.
Dry Ice
1. Excise the tissue from the animal and place in a microfuge tube.
2. Place tube on dry ice with ethanol for 15 minutes.
3. Store at -80oC.
For Organoids and Very Small Tissues (<10mm3)
Organoids and very small tissues (eg. pancreatic islets, embryonic tissues, tissues onlyseveral cell layers thick such as retina or epidermis) should be cryopreserved in 500 μL ofcryopreservation solution – 50% FBS/40% growth media/10% DMSO as if they were cells,
aiming for 100,000 cells total.

*Our Tn5 Transposase assisted chromatin tagmentation methods are covered by thesepatents: US9938524, US10689643B2, EP2783001B1, EP2999784B1.

North America 877 222 9543

Europe +32 (0)2 653 0001
www.activemotif.com
CUT&Tag-IT™ Service Sample Preparation Protocol

Japan +81 (0)3 5225 3638
Rev G091124