CUT&Tag PCR Master Mix #63228

CUT&Tag PCR Master Mix

The CUT&Tag PCR Master Mix is an optimized 2X reaction mix for PCR amplification and DNA library preparation for CUT&Tag DNA samples. The non-hot start DNA polymerase in this product ensures the success of gap filling extension for tagmentated DNA. This master mix formulation is supplied at 2X concentration and contains all PCR reaction components required for amplification of DNA, except primers and the tagmented DNA sample. This product is provided in 840 µL volumes sufficient for preparation of 24 PCR reactions and is compatible with CUT&Tag DNA samples generated by CUT&Tag pAG-Tn5 (Loaded) #79561 or DNA samples from other tagmentation assays, such as ATAC-seq. This product is recommended to be used with the index primers provided in the CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. Alternatively, other tagmentation PCR amplification indexes designed for downstream sequencing with Illumina Systems can be used. This product should not be used for library preparation of DNA samples from ChIP or CUT&RUN assays.

Compatible Reagents:

1. CUT&Tag pAG-Tn5 (Loaded) #79561
2. CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415

Non-Compatible Assay kit:

1. DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795

Required Reagents

Reagents Included:

1. CUT&Tag PCR Master Mix #63228

Reagents Not Included:

1. Forward and Reverse PCR indexes for tagmentated DNA
2. PCR tubes or plate and PCR Machine
3. AMPure XP Beads (Beckman Coulter, Inc. #A63881) or SPRIselect Reagent Kit (Beckman Coulter, Inc. #B23317)
4. 80% Ethanol (freshly prepared)
5. 10 mM Tris-HCl (pH 8.0-8.5)
6. Magnetic rack/stand
7. Agilent Bioanalyzer system and Agilent High Sensitivity DNA Kit (5067-4626)

CUT&Tag PCR Master Mix Protocol

I. Set up the PCR Reaction

Before starting:

• Thaw forward and reverse PCR indexes and CUT&Tag DNA (or any tagmentated DNA) at room temperature. Quick spin to collect all liquid from the sides of the tube.

1. Add the following components to a sterile PCR tube or single well of a PCR plate. Record the CUT&Tag Index 7 added to each PCR tube or well.

   Reagents	Volume for 1 PCR Reaction (70 µl)
   CUT&Tag DNA (or any tagmentated DNA)	30 µl
   CUT&Tag PCR Master Mix	35 µl
   CUT&Tag Index 7 Primer for Illumina Systems (10 µM)	2.5 µl
   CUT&Tag Index 5 Primer for Illumina Systems (10 µM)	2.5 µl

   NOTE: It is critical to change tips between tubes to avoid cross-contamination. If starting with less than 30 µl of DNA template, add DNAse-free water to bring the volume up to 30 µl.

2. Thoroughly mix the reaction by pipetting up and down and perform a quick spin to collect all liquid from the sides of the tube or plate well.

NOTE: It is critical to change tips between samples to avoid cross-contamination.

3. Place the tube on a thermocycler with a heated lid and perform PCR amplification using the following PCR cycling conditions:

   a.	Gap Filling	58°C for 5 min
   b.	Gap Filling Extension	72°C for 5 min
   c.	Initial Denaturation	98°C for 30 sec
   d.	Denaturation	98°C for 10 sec
   e.	Anneal and Extension	60°C for 11 sec
   	For between 20,000 and 100,000 cells per CUT&Tag reaction, repeat steps d and e for a total of 13 cycles.
   	For 20,000 and less cells per CUT&Tag reaction, repeat steps d and e for a total of 14-16 cycles.
   	Note: excessive PCR cycles lead to lower library diversity and/or higher duplication rate of NGS reads.
   f.	Final Extension	72°C for 1 min
   g.	Hold	4°C

4. Proceed to Cleanup of PCR Amplification (Section II). (Safe Stop). Alternatively, samples can be stored at -20°C.

II. Cleanup of PCR Amplification

Before starting:

• If using AMPure XP beads, allow the beads to warm to room temperature for at least 30 minutes before use.
• Resuspend AMPure XP Beads or SPRIselect beads by tube inversion or pipetting up and down.
• Prepare 400 µl of 80% ethanol for each sample.
• Prepare approximately 20 µl of 10 mM Tris-HCl (pH 8.0-8.5) for each sample.

1. Add 70 µl (1.0X) resuspended AMPure XP beads or SPRIselect beads to 70 µl PCR reaction from Step 3 in Section I. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix.
2. Incubate samples on bench top for at least 5 minutes at room temperature.
3. Place the tube/plate on an appropriate magnetic stand for 5 minutes to separate the beads from the supernatant.
4. Carefully remove and discard the supernatant. Be careful to remove all liquid residues but not to disturb the beads that contain DNA targets.
5. Add 200 µl freshly prepared 80% ethanol to the tube/plate while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
6. Repeat Step 5 once for a total of two washes. Be sure to remove all visible liquid after the second wash.
7. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.

NOTE: Do not over-dry the beads. This may result in lower recovery of DNA targets. Elute the samples when the beads are still glossy looking, but when all visible liquid has evaporated. If the beads start to crack, they are too dry.

8. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 17 µl of 10 mM Tris-HCl (pH 8.0-8.5) per sample. Mix well by pipetting up and down 10 times. Incubate for at least 2 minutes at room temperature.
9. Place the tube/plate on the magnetic stand and wait for 5 minutes. Carefully transfer 15 µl of supernatant containing the DNA targets to a new tube. (Safe Stop) DNA libraries can be stored at -20°C until further use.
10. Measure the concentration of library DNA by Nanodrop or Picogreen assay. The concentration of the DNA library should be in the range of 10-40 ng/µl.
11. Dilute 1 µl of the library DNA with 10mM Tris-HCl (pH 8.0-8.5) to a final concentration of 5-10 ng/µl. Use the diluted library DNA to determine size distribution using an Agilent Bioanalyzer High Sensitivity DNA chip, according to the manufacturer's instructions.

NOTE: While CUT&Tag DNA libraries generated for histone modifications typically show robust signal in Bioanalyzer or TapeStation systems analysis, libraries generated for non-histone proteins such as transcription factors and cofactors often have very weak or even no visible signal using Bioanalyzer or TapeStation systems, but still generate NG-sequencing results with high mapping rates, high numbers of identified binding peaks, and acceptable signal-to-noise ratios across the whole genome. Therefore, we recommend sequencing DNA library preps from transcription factor and cofactor CUT&Tag reactions that do not show a signal in Bioanalyzer or TapeStation systems analysis.

12. Dilute final purified library samples with 10mM Tris-HCl (pH 8.0-8.5) for high throughput sequencing. Refer to Illumina sequencing manual for optimal concentration and volume of library DNA required for NG-seq.