Enabling Epigenetics Research

5hmC Profiling Kit
Catalog No. 55023(Version A1)

Copyright 2023 Active Motif, Inc.

Information in this manual is subject to change without notice and does not constitute a
commitment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any
warranty of any kind, either explicit or implied. Information may be changed or updated inthis manual at any time.
This documentation may not be copied, transferred, reproduced, disclosed, or duplicated,
in whole or in part, without the prior written consent of Active Motif, Inc. This documentation
is proprietary information and protected by the copyright laws of the United States andinternational treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2023 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008. All
rights reserved.
All trademarks, trade names, service marks or logos referenced herein belong to theirrespective companies.
For research use only. Not for use in diagnostic procedures.

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Contents
Overview  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Flowchart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  2
Kit Components and Storage  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  3
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4
Protocols  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  5
Buffer Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sample Input Preparation  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  55hmC Profiling Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  8
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section A. Use of Included Control Spike-In DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section B. qPCR recommendations on the Spike-In DNA pool . . . . . . . . . . . . . . . . 15
Section C. % Input Calculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

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Overview
The 5hmC Profiling Kit is designed to detect and capture DNA fragments containing
5-hydroxymethylcytosine (5hmC) for use in genome-wide analysis. The kit works withdouble-stranded DNA that has been fragmented to a size range of 100-500 base pairs. The
DNA is then incubated in the presence of a β-glucosyltransferase enzyme and modified
UDP-glucose donor. The enzyme transfers glucose to 5-hydroxymethylcytosine residues,
creating glucosyl-hydroxymethylcytosine. A biotin conjugate is then chemically attached tothe modified glucose. Next, magnetic streptavidin beads are used to capture the biotinylated
5hmC DNA fragments. Because the spacer between the biotin and glucose moieties contains
a disulfide bond, fragments can be eluted with a reducing agent (elution buffer), therefore
enriching fragments containing 5hmC. Following purification with SPRI beads, the enriched
DNA can be used in qPCR and/or Next-Generation sequencing for targeted or genome-wide
5-hydroxymethylcytosine analysis, respectively.
By utilizing chemical labeling of 5hmC residues, the 5hmC Profiling Kit is extremely specificand sensitive in its capture of hydroxymethylated DNA fragments. The biotin-streptavidin
binding reaction also allows for more stringent binding and wash conditions, enabling
sensitivity and specificity that cannot be detected by antibody immunoprecipitation methods.
Active Motif’s fast, magnetic protocol has been streamlined to minimize the number of wash
and incubation steps, delivering enriched 5hmC DNA in less than 5 hours. For added
convenience, the kit also includes optional Spike-In DNA containing an equimolar mixture of
two DNA fragments containing a small percentage of either 5-hydroxymethylcytosine or
5-methylcytosine. PCR primers targeting these fragments can be used to verify both theefficiency and specificity of the enrichment.
Product

FormatCatalog No.

5hmC Profiling Kit

24 rxns55023

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1Flowchart of Process

1 hourβ-glucosyltransferase
UDP-Azide-glucose

1 hour

Biotin conjugate

1 hourStreptavidin beads

45 minutes

Washing & Elution

qPCR

Sequencing

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Kit Components and Storage
Upon receipt, please store each component at the temperature indicated in the table below.
Kit and components are guaranteed for 6 months after receipt when stored properly.
Kit Component

Quantity

Storage

Beta-glucosyltransferase

55 µL

-20°C

UDP-Azide-Glucose

65 µL

-20°C

Biotin Conjugate Solution

500 µL-20°C

Spike-In DNA (10 ng/µL)

55 µL

-20°C

5hmC Spike-In Primer

160 µL-20°C

5mC Spike-In Primer

160 µL-20°C

10X Reaction Buffer

140 µL

4°C

Streptavidin Beads*

620 µL

4°C

Binding Buffer AM13

28 mL

4°C

10X Elution Buffer AM2

140 µL

4°C

SPRI Beads*

4.5 mL

4°C

TT Buffer

3.6 mL

RT

11 mL

RT

10 mM Tris-HCl, pH 8

* Do not freeze the Streptavidin Beads or the SPRI Beads. Upon receipt of this kit, store thesebeads at 4°C.

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Additional Materials Required• Sample DNA that has been fragmented to between 100-500 bp in size
• End-over-end tube rotator• 80% Ethanol (make fresh)• 0.2 mL PCR tubes or 0.2 mL strip tubes• Magnetic rack for 0.2 mL PCR tubes• PCR tube centrifuge• Low nucleic acid binding 1.5 or 2 mL microcentrifuge tubes (such as Costar
Product no. 3207)• Water bath or Heat block set at 37°C
• DNase-free water

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Protocols
Buffer Preparation
10X Elution Buffer AM2
Prepare a 1X Elution Buffer by diluting the 10X Elution Buffer AM2 in Binding Buffer AM13.
For every 50 µL elution reaction, dilute 5 µL 10X Elution Buffer AM2 into 45 µL Binding
Buffer AM13.
SPRI Beads
Bring to room temperature for at least 30 minutes before use. Vortex thoroughly 10 seconds.

Sample Input Preparation
Sample DNA Input Quantity
To ensure enough enriched DNA for library preparation we suggest using an input of 80
ng – 2.5 µg fragmented genomic DNA. It may be possible to use less DNA, dependingon the sample type. However, with input less than 80 ng, there may be large numbersof PCR duplicates that arise during library preparation making it difficult to accuratelyinterpret sequencing data. The usage of unique molecular indexes (UMIs) may alleviate
the PCR duplication problem by adding unique molecular tags to each molecule before
library amplification.
For tissues with very low levels of 5hmC, such as cancer cells, it may be necessary to use
more than 2.5 µg of DNA. In this case we suggest setting up multiple enrichment reactions
and pooling the DNA together during purification.
Fragments should range in size from 100-500 bp. Use the recommended protocols for
preparing fragmented DNA by restriction enzyme digestion or sonication prior to starting the
5hmC Profiling Kit assay.
Fragmentation of Genomic DNA
Prior to starting the 5hmC Profiling Kit assay, genomic DNA should be fragmented using
either mechanical fragmentation (e.g. sonication) or restriction digestion with a methylationinsensitive restriction enzyme to yield fragments ranging in size from 100 to 500 base pairs.
We recommend preparing high-quality genomic DNA using a commercially available kit or astandard established protocol. The quality of the genomic DNA can be assessed by agarose
gel electrophoresis and DNA concentration can be determined by UV spectrophotometry.
Restriction Digest
Restriction digestion can be used to fragment the genomic DNA by cutting with amethylation-insensitive restriction enzyme. This fragment should be long enough (75 bp or
longer) to allow for PCR analysis. Restriction enzyme digestion is recommended if working
with limited sample amounts due to the smaller volumes that may be used. Restriction
enzyme digestion also offers a more reproducible fragmentation pattern. Some useful
methylation-insensitive restriction enzymes are shown in the below table. As might be

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expected, the enzymes whose recognition sites contain G and C bases cut more frequently in
CpG islands than enzymes whose sites are composed only of A and T bases.
Restriction
EnzymeRecognition
Sequence

No. of fragments (per kb)
in CpG Islands

No. of fragments (per kb)
in non-CpG Islands

Mse I

TTAA

0.8

2.88

Bfa I

CTAG

1.56

1.55

Tas I

AATT

0.8

2.88

Csp6 I

GTAC

2.23

1.41

Follow the recommendations of the enzyme provider to set up the restriction digestion. The
following is an example protocol. This protocol can be modified depending on the amount ofisolated genomic DNA or the restriction enzyme being used. The example below
demonstrates a restriction digestion of 1 µg DNA with Mse I (New England Biolabs (NEB)).
Scale up the reaction as needed for larger DNA quantities.
Genomic DNA

1 µg

10X rCUTSmart Buffer

5 µL

Mse I (10 U/µL)

1 µL

dH2O
Total volume

to 50 µL
final volume
50 µL

1.

Mix well by pipetting and incubate at 37°C for 1 hour to overnight.

2.

Heat-inactivate Mse I by incubating the reaction mixture at 65°C for 20 minutes.

3.

Purify the DNA using either phenol/chloroform extraction and precipitation or using acommercial DNA Purification Kit such as the QIAGEN MinElute Reaction Cleanup Kit(Cat No. 28204).

Mechanical Fragmentation (sonication)
Mechanical fragmentation is ideal when using a large quantity of genomic DNA. In general,the DNA should be sheared to an average fragment size of less than 500 bp. A multi-sample
sonicator like Active Motif’s PIXUL® or a probe sonicator like Active Motif’s EpiShear™ can beused for that purpose.
A protocol optimized with the EpiShear™ is shown below.
1.

Transfer genomic DNA into a 1.5 mL microcentrifuge tube and adjust final volume to
300 µL by addition of 10 mM Tris-HCl, pH 8. We suggest using 20 µg or more DNA
per sonication reaction.

2.

Using a tip probe sonicator, sonicate on ice with 6 pulses of 30 seconds “on” (45%amplitude if using Active Motif’s EpiShear™ sonicator), and a 30-second pause on ice
between each pulse. The sheared DNA can be visualized by electrophoresis on a 2%
agarose gel.
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6Spike-In DNA (10 ng/µL)
The optional positive control Spike-In DNA (10 ng/ µL) is meant to be added to thefragmented sample DNA before adding to the glucosylation reaction, in order to verify
efficient and selective capture of 5hmC. We recommend maintaining a ratio of Spike-In
DNA to fragmented sample DNA of 1:10,000 (e.g., 2.5 ng Spike-In DNA (10 ng/ µL) in 25 µg
fragmented genomic DNA = 1:10,000 ratio). For more detailed instructions, please refer to the
Appendix, Section A. Use of Included Spike-In DNA on page 13.
The Spike-In DNA (10 ng/µL) contains an equimolar mixture of two different dsDNA
fragments which have been amplified using a small admixture of either 5-methylcytosinesor 5-hydroxymethylcytosines.
These fragments were separately amplified from the rabbit IgH locus, pooled at equimolar
concentrations, and stored at 10 ng/µL. They should be diluted just before use and the stock
solution should always be kept frozen.
The two Spike-In DNA fragment sequences are shown below.
5mC DNA fragment 266 bp sequence:
5’TTCTCTCCACAGGTGTCCACTCCCAGGTCCAAGTTTAAACTCTAGGAAGGAGGGCCACCATGACCGAGTGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTACGAATTCGCAGTCGGTGGAGGAGTCCGAGGGAGGCCTGATCAGGCCTGGAGGAACCCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTAGGTATGGAGTGACCTGGGTCCGCCAGGCTCCAGGGAACGGGCTGGAATGGATCGGATTC3’5hmC DNA fragment 207 bp sequence:
5’TTGCCTTTCTCTCCACAGGTGTCCACTCCCAGGTCCAAGTTTAAACTCTAGGAAGGAGGGCCACCATGACCGAGTGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTACGAATTCGGCCCTTGTGATGACCCAGACTCCACATTCCACGTCTGCGGCCGTGGGAGGCACAGTCACCATCAACTGCCAGTCCAGTCAGA3’

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75hmC Profiling Protocol
Glucosylation Reaction (60 minutes)
1.

Calculate the volumes of reagents needed for each glucosylation reaction using thetable below.

2.

Set up a 200 µL PCR tube for each glycosylation reaction to be performed.
Sample: The sample reactions will contain all reagents required for the betaglucosyltransferase to selectively transfer glucose to 5hmC in the DNA fragments.
Negative Control: We recommend a negative control reaction alongside thesamples which does not contain the UDP-Azide-Glucose donor. There should be noenrichment of 5hmC in the negative control.
Fragmented Sample DNA + Spike-In DNA*: The recommended range for fragmented
sample DNA is between 0.08 µg and 2.5 µg. We suggest a starting quantity of 2.5 µg
per sample. For instructions on adding the optional Spike-In DNA to the fragmented
sample please see the Appendix, Section A. Use of the Included Spike-In DNA on
page 13.

Note:

The combined total of Spike-In DNA + fragmented sample DNA should notexceed 40 µL.

Sterile water: Determine the amount of sterile water needed to bring the reaction toa final volume of 50 µL.
3.

Add reagents in the order listed below to each PCR tube.
Reagents

SampleNegative Control

Sterile water____ µL____ µL

10X Reaction Buffer

5 µL

5 µL

UDP-Azide-Glucose

2.5 µL–____ µL____ µL

Beta-glucosyltransferase

2 µL

2 µL

Total Volume

50 µL

50 µL

Fragmented sample DNA + Spike-In DNA**The Spike-In DNA is optional.
4.

Pipette up and down to ensure complete mixture. Cap the PCR tubes tightly. Quickly
spin the tubes to collect the material to the bottom.

5.

Incubate the reaction at 37°C for 1 hour.

Biotinylation Reaction (60 minutes)
6.

Quickly spin the PCR tubes to collect the contents at the bottom.

7.

Add 20 µL Biotin Conjugation Solution to each reaction.

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8.

Cap the PCR tubes tightly. Vortex the reaction to ensure complete mixture. Quickly
spin the tubes to collect the material to the bottom.

9.

Incubate the reaction at 37°C for 1 hour.

Purification of Biotinylated DNA (30 minutes)
Perform SPRI clean-up of biotinylated DNA using 105 µL SPRI bead solution, 1.5X thesample volume.
Note:

The SPRI beads should be brought to room temperature for 30 minutes andthen vortexed for 10 seconds before using.

10. Add 105 µL of well-mixed, room temperature SPRI beads to each tube containing thebiotinylated DNA.
11. Pipet up and down 5-10 times.
12. Incubate the tubes for 5 minutes.
13. Place on magnet to collect beads for 10 minutes.
14. Aspirate the supernatant and discard.
15. Repeat the following wash step 2 times.
a. With the magnet still applied, add 200 µL of freshly prepared 80% ethanol.
b. Incubate for 30 seconds at room temperature.
c. Aspirate the supernatant and discard.
16. Allow tubes to sit at room temperature so that residual ethanol can evaporate. Oncethe beads transition from shiny to matte (2-5 minutes) proceed to the next step.
17. With the tubes off the magnet, resuspend the beads using 55 µL of 10 mM Tris HCl,
pH 8.0. Wait 2 minutes.
18. Place the tubes on the magnet for 2 minutes.
19. For future sequencing or qPCR analysis, a small amount of the supernatant will be put
aside as Input and stored at -20°C by doing the following:a. Transfer 5 µL of the supernatant into a clean PCR tube.
b. To this add 15 µL of TT Buffer for a final volume of 20 µL.
Note:

The TT Buffer contains 0.05% Tween-20 to minimize the loss of DNA.

c. Store this DNA at -20 °C.
20. Pipette off the remaining supernatant (~50 µL) from the magnetic beads into a second
clean PCR tube.
21. This DNA can be safely stored in tubes at -20°C until use in the capture reaction.

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9Capture Reaction (60 minutes)
Be sure the Streptavidin Beads are fully resuspended before adding to each capture reaction
by vortexing. If working with multiple reactions, the beads may need to be resuspended morethan once.
22. In a fresh set of 0.2 mL PCR tubes prepare the following reactions:
Reagents

SampleNegative Control

Streptavidin Beads

25 µL

25 µL

Binding Buffer AM13

25 µL

25 µL

Purified Biotinylation Reaction

50 µL

50 µL

Total Volume

100 µL100 µL

23. Incubate the reactions for 1 hour at room temperature with end-to-end rotation.
Washing and Elution (60 minutes)
Before washing and elution be sure that you’ve prepared 1X Elution Buffer by diluting the 10X
Elution Buffer AM2 into Binding Buffer AM13. For every 50 µL elution reaction, dilute 5µL 10X
Elution Buffer AM2 into 45 µL Binding Buffer AM13.
24. After the capture step is complete, spin the PCR tubes briefly and place tubes ona magnetic stand to pellet beads to the side of the tube. If further analysis of theunbound fraction will be performed, place supernatant in a microcentrifuge tube andstore at -20°C. Otherwise, carefully remove and discard the supernatant.
25. Repeat the following wash steps (a-c) five times:
a. Add 200 µL Binding Buffer. Pipette 2-3 times gently to resuspend. Ensure that thebeads do not stick to the pipette tips. [Depending on the strength of the magnet
being used, it may be necessary to remove the tubes from the magnet and place
in a separate rack to fully resuspend the beads.]b. Place tubes on magnetic stand and allow beads to pellet on the side of the tube.
c. Carefully remove the supernatant and any residual bubbles.
26. After all five washes resuspend the beads with 50 µL 1X Elution Buffer by pipetting
2-3 times.
27. Incubate for 30 minutes at room temperature with end-to-end rotation.
28. Briefly centrifuge the tubes to collect liquid from the cap.
29. Place tubes in magnetic stand and allow beads to pellet onto tube sides.
30. Transfer the supernatant, which contains the enriched DNA, to a fresh tube.
31. Proceed to Purification of Enriched DNA.

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Purification of Enriched DNA (30 minutes)
Perform SPRI clean-up of enriched DNA using 75 µL SPRI bead solution, 1.5X thesample volume.
Note:

The SPRI beads should be brought to room temperature for 30 minutes andthen vortexed for 10 seconds before using.

32. Add 75 µL of well-mixed, room temperature SPRI beads to each tube containing thebiotinylated DNA.
33. Pipet up and down 5-10 times.
34. Incubate the tubes for 5 minutes.
35. Place on magnet to collect beads for 10 minutes.
36. Aspirate the supernatant and discard.
37. Repeat the following wash steps 2 times.
a. With the magnet still applied, add 200 µL of freshly prepared 80% ethanol.
b. Incubate for 30 seconds at room temperature.
c. Aspirate the supernatant and discard.
38. Allow tubes to sit at room temperature so that residual ethanol can evaporate. Oncethe beads transition from shiny to matte (2-5 minutes) proceed to the next step.
39. With the tubes off the magnet, resuspend the beads using 20 µL of TT Buffer. Wait
2 minutes.
40. Place the tubes on the magnet for 2 minutes.
41. Pipette the supernatant from the magnetic beads into a clean PCR tube.
42. For long term storage transfer to a low nucleic acid binding tube at -20 °C until use indownstream analysis.
Downstream Analysis
1.

Quantify the 5hmC enriched DNA using a fluorescent quantitation system such as a
Qubit. Depending on the level of 5hmC in the sample, Nanodrop or other quantitation
systems may not be able to accurately quantify the DNA. Verify that the sample falls
within the detectable range of the quantification method selected.

2.

If desired, perform real time PCR analysis as a check for enrichment before library
preparation. We suggest using 2µL of pulldown for qPCR and 18 µL of pulldown
for library construction. If the Optional Spike-In DNA was used, run qPCR with theprovided Primer Mix according to the instructions in the Appendix, Section A. Use of
the Included Control Spike-In DNA on page 13 in order to determine if enrichment
was successful. Alternatively, you could amplify your own targets of interest in the
DNA samples. The amplification conditions will need to be optimized for each target
locus, master mix, and instrument.

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3.

Follow the recommendation for library preparation based on the sequencing platform
to be used. This process involves adding adapters to the ends of the dsDNA
fragments. The library is then PCR amplified and validated prior to sequencing.
Use a commercially available library preparation kit and follow the manufacturer’s
recommendations. Enriched DNA from Active Motif’s 5hmC Profiling Kit has been
extensively validated using the Illumina® sequencing platforms. The Input DNA set
aside can also be submitted for sequencing to determine the baseline peaks foreach sample.

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Appendix
Section A. Use of Included Control Spike-In DNA
A control containing both 5-methylated (5mC) and 5-hydroxymethylated (5hmC) DNA isincluded as an optional validation of the enrichment efficiency and specificity. This SpikeIn DNA is provided at 10 ng/µL. When the Spike-In DNA is added to the sample DNA, thefragments containing 5hmC will undergo the glycosylation and biotinylation reactions andwill be captured by the streptavidin beads. In contrast, the 5mC containing fragments will
not undergo these reactions and provide only background signal. Following elution andpurification, the DNA can be analyzed using real time PCR with the provided primer mix toverify the efficiency of the capture method. Because the fragment sequences in the Spike-In
DNA come from rabbit genomic DNA, they will not interfere with capture reactions on humanand mouse genomic DNA.
We recommend using a 1:10,000 ratio of Spike-In DNA (ng) to fragmented sample DNA (ng)for each glycosylation reaction. The Spike-In DNA will need to be pre-diluted first before
adding to the fragmented sample DNA. For convenience below are some recommendations
for pre-dilution of the Spike-In DNA for different sample ranges.
Input is 20 – 200 ng fragmented sample DNA
1.

First create a 1:10,000 diluted stock of Spike-In DNA.
a. Label three 0.2 mL PCR tubes 1:100, 1:1,000, and 1:10,000 respectively.
b. Into the 1:100 tube, dispense 198 µL 10mM Tris HCl, pH 8 and 2 µL of Spike-In DNA(10 ng/µL).
c. Vortex tube for 3 seconds.
d. Into the 1:1000 tube, dispense 90 µL 10mM Tris HCl, pH 8 and 10 µL of theprevious 1:100 dilution.
e. Vortex tube for 3 seconds.
f. Into the 1:10,000 tube, dispense 90 µL 10mM Tris HCl, pH 8 and 10 µL of theprevious 1:1,000 dilution.
g. Vortex tube for 3 seconds.
h. This tube now contains Spike-In DNA at a concentration of 0.001 ng/µL.

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2.

The equation below can be used to calculate the volume (V) of this diluted Spike-In
DNA (0.001 ng/µL) to add to the sample DNA (S) to create a 1:10,000 ratio of Spike-In
DNA to sample DNA:S
10,000V=
0.001 ng/µL
For example, if using 25 ng fragmented sample DNA (S) in the glycosylation reaction
we recommend adding 2.5 µL of diluted (0.001 ng/µL) Spike-In DNA (2.5 pg) for a
1:10,000 ratio.

Note:

The total volume of Spike-In DNA + fragmented sample DNA should notexceed 40 µL per glycosylation reaction.

Input is 200 – 2000 ng fragmented sample DNA
1.

First create a 1:1,000 diluted stock of Spike-In DNA.
a. Label two 0.2 mL PCR tubes 1:100 and 1:1,000 respectively.
b. Into the 1:100 tube, dispense 198 µL 10mM Tris HCl, pH 8 and 2 µL of Spike-In DNA(10 ng/µL).
c. Vortex tube for 3 seconds.
d. Spin down the liquid for 3 seconds with benchtop minicentrifuge. This tube now
contains Spike-In DNA at a concentration of 0.1 ng/µL.
e. Into the 1:1000 tube, dispense 90 µL 10mM Tris HCl, pH 8 and 10 µL of theprevious 1:100 dilution.
f. Vortex tube for 3 seconds.
g. Spin down the liquid for 3 seconds with benchtop minicentrifuge. This tube now
contains Spike-In DNA at a concentration of 0.01 ng/µL.

2.

The equation below can be used to calculate the volume (V) of this diluted Spike-In
DNA (0.01 ng/µL) to add to the sample DNA (S) to create a 1:10,000 ratio of Spike-In
DNA to sample DNA:S
10,000V=
0.01 ng/µL
For example, if using 250 ng fragmented sample DNA (S) in the glycosylation reaction
we recommend adding 2.5 µL of diluted (0.01 ng/µL) Spike-In DNA (25 pg) for a
1:10,000 ratio.

Note:

The total volume of Spike-In DNA + fragmented sample DNA should notexceed 40 µL per glycosylation reaction.

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14Section B. qPCR recommendations on the Spike-In DNA pool
The Spike-In DNA (10 ng/µL) contains an equimolar mixture of two different dsDNA
fragments which have been amplified using a small admixture of either 5-methylcytosinesor 5-hydroxymethylcytosines. Post-assay enrichment efficiency before proceeding tolibrary preparation and sequencing can be determined with qPCR performed on these
Spike-In targets.
To use the Spike-In QC in conjunction with generated libraries for sequencing, follow the
protocol below. You’ll be using 10% (by volume) of the Input and enriched pulldown material
for qPCR analysis. Use the remaining material for library construction.
Note:

It is recommended to prepare triplicates of each sample and Input reaction.

1.

Add 48 µL TT Buffer into a PCR tube along with 2 µL of the 20 µL Input DNA (setaside in after Purification of Biotinylated DNA, Step 19).

2.

Vortex and spin down.

3.

Add 48 µL of TT Buffer into a PCR tube along with 2 µL of the 20 µL enriched DNA(from Purification of Enriched DNA, Step 42).

4.

Vortex and spin down.

5.

Assemble a master mix for the number of samples required, for either the 5mC or the
5hmC Primer Mix, according to the reaction table below.
Reagent

20 µL PCR reactions

Fast SYBR Green master mix

10 µL

5mC or 5hmC Primer Mix*

2 µL

Sterile water

3 µL

DNA sample (eluted or Input)

5 µL

Total Volume

20 µL*The provided PCR Primer Mixes contain both Forward and Reverse primers for use
with the provided control DNA. Use 2 µL of the appropriate PCR Primer Mix (either
5mC or 5hmC) in the 20 µL reaction for the PCR protocol described above.
6.

Place tubes in a Real Time PCR instrument and program as shown below. The
amplification conditions should be optimized for each target locus, master mix
reagent and PCR instrument. A suggested starting point is:
Step

Protocol1

95 °C for 2 minutes

2

95 °C for 10 seconds

3

65 °C for 30 seconds

Repeat 40x

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15Section C. % Input Calculation
To calculate % Input:
Take the average of the PCR triplicate CTs (n=3) together for each sample. Subtract -3.32
from Input CT value to correct for input amount relative to its pulldown. Use the following
calculation for % Inputs per target:% input = (2–(Avg Pulldown Ct-Avg Input Ct)) x 100
Our suggested passing criteria for the Spike-In DNA:
% Input 5hmC >9.5%% Input 5mC <1%
Below is example data for Spike-In DNA enrichment. In this experiment 31 pg of Spike-In DNA
was added to 310 ng of mouse brain gDNA and run through the 5hmC Profiling Kit protocolin triplicate. RT-PCR was done using either the 5mC or the 5hmC primer mix for both the
Input and the post-enrichment DNA. The % Input was calculated using the average CT values
for each reaction and plotted below. The enrichment was 13.52 for 5hmC and 0.02 for 5mC.

% Input for Spike-in DNA
16
14% Input

12
10
8
6
4
2
0

5hmC

5mC

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16Troubleshooting Guide
Problem/Question

Recommendation

Little or no enrichment ofmethylated DNA

The 5hmC Profiling Kit is optimized for use with 0.08 – 2.5 µg offragmented (<500 bp) DNA per reaction. For samples with low levels of
5hmC it may be necessary to use more sample material per reaction.
If using more than 2.5 µg, set up multiple reactions and pool samples
together during the Purification of Enriched DNA.
Make sure reagents were prepared as stated in the Buffer Preparation
and Recommendation section prior to starting the assay. Reagentsshould be added in the order listed.

Should I use Restriction Digest
or Sonication to fragment
my DNA?

Restriction Digest is very precise and reproducible; however, theDNA must be well purified, and analysis of several loci may also
require use of different enzymes. In addition, the region of interest
may not be flanked by suitable restriction sites and single-nucleotide
polymorphisms (SNPs) between different cell types may confoundresults. In contrast, Sonication is random, which enables analysis ofmany loci simultaneously.

Should I use heat inactivation toremove the restriction enzymes
used to fragment DNA?

After restriction digest, we recommend that samples be treated for 20
minutes at 65°C. Some enzymes (such as Mse I) will be inactivated bythis treatment, while those that are not will be forced off the DNA. In
most cases (even when using enzymes that are not heat-inactivated),
DNA treated in this fashion should be suitable for use in the 5hmC
Profiling Kit protocols. In some situations (e.g., when the DNA used ina digest is contaminated with cellular proteins or when a large amountof restriction enzyme is required for the digest) it may be desirable
to purify the digested DNA by purification columns or through phenol
extraction/ethanol precipitation.

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China

Japan

EuropeNorth America

If you need assistance at any time, please call or send an e-mail to Active Motif Technical
Service at one of the locations listed below.

Toll free:877 222 9543

Direct:

760 431 1263

Fax:

760 431 1351

E-mail:tech_service@activemotif.com

UK Free Phone:

0800/169 31 47

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0800/90 99 79

Germany Free Phone:

0800/181 99 10

Direct:+32 (0)2 653 0001

Fax:

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E-mail:eurotech@activemotif.com

Direct:+81 (0)3 5225 3638

Fax:

+81 (0)3 5261 8733

E-mail:japantech@activemotif.com

Direct:(86)-21-20926090

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techchina@activemotif.com

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