Low Cell ChIP Kit(version A1)
Catalog No. 53086

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 in this manual at any
time.
This documentation may not be copied, transferred, reproduced, disclosed, or duplicated, inwhole 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 and international
treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2022 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 their respective
companies.

www.activemotif.com

TABLE OF CONTENTS

Page

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Flow Chart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Protocol Overview and Time Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Kit Components and Storage
Low Cell ChIP Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Protocol 1 - Low Cell ChIP
Cell Growth and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Buffer Preparation & Recommendations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Section A. Cell Fixation Starting with Cultured Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Section B. Cell Fixation Starting with Small Tissue Biopsies . . . . . . . . . . . . . . . . . . . . 12
Section C. Chromatin Sonication. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section D. Protein G Agarose Bead Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section E. Pre-clearing the Chromatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
Section F. Immunoprecipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Section G. Washing & Elution of IP Reactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Section H. Reversal of Cross-links and DNA Purification . . . . . . . . . . . . . . . . . . . . . . . .17
Section I. Troubleshooting Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

www.activemotif.com

Overview
Chromatin Immunoprecipitation (ChIP) is a powerful tool for studying protein/DNA interactions,
including transcription factors, co-regulatory proteins, modified histones, chromatin-modifying
enzymes and polymerases because it enables identification of the localization of proteins boundto specific DNA loci. ChIP-Seq analysis typically requires millions of cells per immunoprecipitation
reaction in order to obtain meaningful information about global changes across a large population of cells. To address the needs of researchers working with limited sample material, or thosewanting to study the complexities of protein-DNA interactions within a small population of cells,
Active Motif has utilized its expertise in ChIP to develop the Low Cell ChIP Kit.
The Low Cell ChIP Kit provides an optimized ChIP workflow to generate genome-wide binding profiles from as few as 1,000 cells, or small tissue biopsies. Low Cell ChIP not only reduces
sample input requirements, but also improves signal-to-noise through the use of low backgroundProtein G agarose beads and blockers.
The Low Cell ChIP Kit contains sufficient reagents to perform 16 ChIP-Seq reactions. Reagents areincluded for chromatin preparation and immunoprecipitation.

product

formatcatalog no.

Low Cell ChIP Kit

16 rxns53086

www.activemotif.com

1Flow Chart of Low Cell ChIP Process

Day 1
Count cultured cells
or small tissue biopsies

Formaldehyde fix cells& sonicate chromatin

Transfer cells tosiliconized tube

3-6 hours

2-3 hours

Pre-clear thechromatin

Day 2 Prepare agarose beads
for pre-clearing reaction&

Add antibody to chromatin

Prepare agarose beads
for IP reaction
Overnight

Overnight

3-4 hours

Day 3

Add IP beads toantibody bound chromatin

Wash antibody boundprotein/DNA complexes
using ChIP filtration columns

Overnight

Day 4

DNA purificationwww.activemotif.com

NGS Library preparation

2

Elution from column &reversal of cross-links

Protocol Overview and Time Table
Protocol Steps

Required Time

Cell Collection and Fixation

30 minutes

Chromatin Sonication*

20 minutes per sample

Protein G Agarose Bead Preparation*3 - 6 hours (Pre-clearing beads) to overnight (IPbeads)

Pre-clearing the Chromatin*

2 - 3 hours

Immunoprecipitation

Overnight incubation

Binding to Protein G Agarose Beads

3 - 4 hours

Wash Immune Complexes & Elute DNA

45 minutes

Reversal of Cross-links

Overnight incubation

DNA Purification

1.5 hours

www.activemotif.com

3Kit Performance and Benefits
Low Cell ChIP-Seq Advantages:• Reproducible ChIP data from as little as 1,000 cells for high abundance target proteins andas little as 50,000 cells for lower abundance transcription factors• Works with cultured cell lines, primary cells, including FACS sorted cells, and small tissue
biopsies• Works with active and repressive histone modifications and transcription factor targets

Low Cell ChIP-Seq Kit

Figure 1: Sensitivity of Low Cell ChIP-Seq Kit across different target proteins and input amounts.
The Low Cell ChIP-Seq Kit was used to detect both robust and low abundance target proteins and both active and repressive
histone modifications using the input cell number listed in the y-axis. Nice peaks are observed above background from as
little as 1,000 cells for histone marks H3K4me3, H3K4me1 and H3K27me3. Lower abundance proteins BRD4 and insulator
protein CTCF show nice ChIP-Seq signal from only 10,000 cells. Results shown represent a collection of individual Low Cell
ChIP-Seq reactions from several cell lines. Data has been reproducibly generated across multiple experiments.

www.activemotif.com

4

Figure 2: Comparison of Low Cell ChIP-Seq data with ENCODE validated data sets.
The Low Cell ChIP-Seq Kit was used to detect histone modifications associated with either active gene expression (H3K27ac)
or repressive chromatin (H3K27me3). Data from Low Cell ChIP-Seq was generated using 5,000 cells per IP reaction, while the
ENCODE data sets were generated from 20 million cells per IP reaction. Results show that Active Motif’s Low Cell ChIP-Seq
Kit is capable of generating high quality ChIP-Seq data that contains the same peak profiles when compared to publisheddata sets.

The quality of the Low Cell ChIP-Seq depends not only on cell type and abundance of the target
protein, but it is also influenced by the quality of the ChIP-Seq antibody. Below is a list of someof the validated antibodies from Active Motif for use with Low Cell ChIP-Seq. Please refer to our
website for an up-to-date list of validated antibodies www.activemotif.com/chip-lowcell.

Protein Target

Active Motif
Catalog Number

Cell Type Tested

H3K4me339159

GM12878; MDA-MB-468

H3K9ac39918

GM12878

H3K9me339161

GM12878

H3K27ac

39133

HepG2

H3K27me3

39155

MDA-MB-468

CTCF

61311

MDA-MB-468

www.activemotif.com

5Kit Components and Storage
The Low Cell ChIP-Seq Kit is for research use only. Not for use in diagnostic procedures. The
kit contains multiple modules to help organize your experiments. Store each component at thetemperatures indicated in the tables below.

Reagents for Low Cell ChIP
Reagents

Quantity

Storage

Proteinase K (10 µg/µl)

80 µl

-20°C

10X PBS

20 ml

-20°C

100 mM PMSF

150 µl-20°C

Protease Inhibitor Cocktail (PIC)

100 µl-20°C

Carrier

35 µl

-20°C

Blocker

100 µl-20°C

Blocking Reagent AM1

115 µl-20°C

BSA (10 mg/ml)

115 µl-20°C

Fixation Buffer

150 µl4°C

Protein G Agarose Beads*

1.2 ml4°C

TE pH 8.0

35 ml

RT

5 M NaCl

400 µlRT

Stop Solution

1 ml

RT

ChIP Filtration Columns

16 ea

RT

ChIP Buffer

45 ml

RT

Wash Buffer AM1

30 ml

RT

LiCl Buffer

40 ml

RT

Elution Buffer AM4

2 ml

RT

1.7 ml siliconized tubes

32 ea

RT

* The Protein G Agarose Beads are shipped on dry ice and can be stored frozen until their firstuse. Once thawed, the Protein G beads should not be re-frozen by the customer. Protein G
Agarose Beads should be stored at 4ºC.

www.activemotif.com

6

Additional materials required• A ChIP-Seq-validated antibody directed against the protein of interest• 37% formaldehyde solution with 10-15% methyl alcohol to prevent polymerization (e.g.
Sigma Aldrich Catalog No. 252549). Do not use paraformaldehyde.
• Phenol, saturated (DNA Purification, Molecular Biology Grade, Amresco Catalog No. 0945)• Chloroform/isoamyl alcohol (24:1) (DNA Purification, Molecular Biology Grade)• 100% ethanol (absolute)• 70% ethanol• DNase-free H2O• Apparatus to rotate tubes end-to-end at 4°C (e.g. a Labquake from Barnstead/Thermolyne
with a tube holder for 1.5 ml microcentrifuge tubes)• Microcentrifuge (table top centrifuge 4°C) and microcentrifuge tubes• 250 µl PCR tubes• Thermal cycler• 15 ml conical tubes• Pipettors and tips (filter tips are recommended)• Sonicator (e.g. Active Motif’s EpiShear™ Sonicator with a 1/8” probe (Catalog No. 53051) with
the EpiShear™ Cooled Sonication Platform (Catalog No. 53080))• (Optional) Enzymes and apparatus to create single cell suspension from tissue samples (e.g.
Disposable Pellet Mixer, VWR 47747-370 or Mini Mortar and Pestle, Bel-Art H37260-0100)

www.activemotif.com

7

PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING!
The Low Cell ChIP-Seq Kit requires multiple days to complete. It is strongly advised to read theentire protocol before starting and to plan your experiments in advance. Please refer to the
kit contents and additional materials required section of the manual (pages 6-7) to identify thecomponents that are needed for each protocol.

Protocol 1 – Low Cell ChIP-Seq
This section describes the experimental set-up and buffer preparation steps needed for the low
cell chromatin preparation and immunoprecipitation portion of the protocol.

Cell Growth Recommendations
When planning an experiment, calculate the number of ChIP reactions you plan to perform. Be
sure to include the appropriate positive and negative control ChIP reactions in your calculations.
Also, note that if you wish to analyze the effect of particular compounds or culturing condition ontranscription factor/DNA interactions, you should prepare chromatin from control (untreated) cells
as a reference sample.
Below are guidelines for cell culturing conditions. If multiple ChIP reactions will be performed
using the same cell type, cells can be cultured in a larger volume and will be transferred to individual tubes for chromatin preparation and immunoprecipitation reactions.
96-wellplate

24-wellplate

12-well plate

6-well plate

T-25 flask

Seeding Density

0.02 x 106

0.05 x 106

0.1 x 106

0.3 x 106

0.7 x 106

Cells at 70-80%
Confluency*

0.06 x 10

0.15 x 10

0.3 x 100.9 x 10

2.1 x 106

Growth Medium
volume to culture
cells

100 µl

1 ml

2 ml

3 ml

5 ml

6

6

6

6

Trypsin

50 µl

500 µl

1 ml

2 ml

3 ml

Growth medium

50 µl

500 µl

1 ml

2 ml

3 ml

1 X PBS(volume per wash)

100 µl

1 ml

2 ml

3 ml

5 ml

Table 1: Cell culture recommendations.
*The number of cells on a confluent plate or dish will vary with cell type. For this table, HeLa cells were used
Please adjust as needed based on your particular cell type.

Recommendations about the number of cells to use per ChIP reaction are provided in the tableon page 11 and are based on the relative abundance of the target protein. ChIP efficiency will also
be influenced by the quality of the ChIP-Seq antibody used. Optimization may be required.

www.activemotif.com

8

Target protein

ExamplesMinimum Cell
NumberRobust histone

H3K4me1, H3K4me3, H3K27me3

1,000 cells

Low abundance histone

H3K27ac, H3K9ac, H3K9me3

5,000 cells

High abundance protein

CTCF, BRD4

10,000 cells

Low abundance protein

Transcription factors

50,000 cells

Buffer Preparation

Complete Cell Fixation Solution
Buffer should be prepared fresh before each experiment. In a 1.5 ml microcentrifuge tube add
62.8 µl sterile water and 7.2 µl fixation buffer. Using appropriate precautions (i.e. safety glasses,
gloves and lab coat), add 30 µl 37% formaldehyde to the tube and vortex to mix. Use 5 µl Complete cell fixation solution per 50 µl cell pellet. Discard unused solution.
Complete Tissue Fixation Solution
Buffer should be prepared fresh before each experiment. In a 1.5 ml microcentrifuge tube add
1 ml 1X PBS. Using appropriate precautions (i.e. safety glasses, gloves and lab coat), add 28 µl
37% formaldehyde to the tube and vortex to mix. Use 1 ml Complete tissue fixation solution per
tissue sample. Discard unused solution.
Stop Solution
Is provided ready to use.
1X PBS Buffer
Prepare a 1X PBS solution by adding 1 ml 10X PBS to 9 ml sterile water. Mix by inverting and place
on ice to chill. 1X PBS Buffer can be prepared in large quantities and stored at 4ºC for 6 months.
100 mM PMSF and Protease Inhibitor Cocktail (PIC)
Thaw the PMSF and the PIC at room temperature until fully dissolved, which takes about
30 minutes. Vortex gently and spin down briefly before use, then add to the buffers immediately
before use.
ChIP Buffer
Is supplied ready to use.
Protein G Agarose Beads
For best results, gently shake and invert the tube to resuspend the agarose beads. The beads
settle quickly, and therefore should be resuspended just before pipetting. We recommend cutting
2 mm from the end of a pipet tip prior to pipetting to prevent the tip from becoming clogged.
Protein G Agarose Beads are shipped on dry ice and can be stored frozen until their firstuse. Once thawed, beads should not be re-frozen by the customer. Protein G Agarose Beads
should be stored at 4ºC.

www.activemotif.com

9

Recommendations
ChIP-Seq-validated Antibody
We recommend using 2-4 µg antibody per ChIP reaction in a maximum volume of 30 µl. However,this will vary according to the affinity of the antibody and the quality of the chromatin; you may
need to use more of a particular antibody. ChIP antibodies must recognize fixed, native proteinthat is bound to DNA and/or complexed with other proteins. Many antibodies that perform well inother applications do not perform in ChIP. Thus, Low Cell ChIP-Seq performed with an antibody
that has not been ChIP-Seq-validated must include appropriate controls. To see a list of available
ChIP-validated antibodies available from Active Motif, please visit www.activemotif.com/chipabs.
Chromatin Shearing Tips
We suggest using a probe sonicator (i.e. Active Motif’s EpiShear Probe Sonicator) which employs adirect sonication method to prepare chromatin for use in the Low Cell ChIP-Seq Kit. Indirect sonication systems may require longer sonication times to achieve optimal chromatin shearing. ChIP
experiments usually require chromatin that has been sheared to a size of 200-1200 bp. In general,
shearing efficiency is improved through the use of a small shearing volume and a V-bottom tube
rather than a round-bottom tube. Also, note that shearing is inefficient if the chromatin sample
becomes emulsified with air bubbles. To determine the appropriate shearing level for your sample,
set up a “practice” tube containing only ChIP Buffer. Slowly increase the sonication amplitude until
foaming starts to occur. Reduce the amplitude setting down slightly and mark this as the highest
possible intensity to use without foaming. If a chromatin preparation becomes emulsified inadvertently, discontinue shearing and centrifuge the sample at maximum speed for 4 minutes at 4ºC ina microcentrifuge to remove trapped air. Finally, to prevent overheating and denaturation of chromatin, samples should be kept on ice as much as possible during shearing, and shearing should be
performed discontinuously (i.e. sonicate for 20 seconds, then place on ice/water for 30 seconds,
sonicate again for 20 seconds, etc.). If possible, shear while on ice or use Active Motif’s EpiShear™
Cooled Sonication Platform (Catalog No. 53080) to help regulate sample temperature.
Safety Precautions
Formaldehyde and PMSF are highly toxic chemicals. Appropriate safety precautions (i.e. safety
glasses, gloves and lab coat) should be used. Also, formaldehyde is highly toxic by inhalation andshould be used only in a ventilated hood. Finally, chromatin sonication should be performed in abiosafety hood if the chromatin is extracted from biohazardous or infectious materials.
Reduce Contamination
To reduce the risk of DNA and library contamination, particularly with ultra-low input samples,
we suggest to physically separate the laboratory space, equipment and supplies where pre-PCR
and post-PCR processes are performed. We also suggest to clean lab areas using 0.5% Sodium
Hypochlorite (10% bleach). The use of specialty barrier pipette tips also helps to avoid exposure
to potential contaminants.

www.activemotif.com

10

Protocols
Section A: Cell Fixation Starting with Cultured Cells
This protocol describes cell fixation and chromatin preparation from cultured cells.
1.

Use the table on page 10 as a guideline to culture each cell line to be tested. Grow the cells
to 70-80% confluency. Stimulate cells as desired to activate the pathway of interest.

2.

Aspirate growth medium and wash the cells twice with the recommended volume of 1X PBS.

3.

Detach cells from plate using trypsin. Transfer cells to a 15 ml conical tube or 1.5 ml microcentrifuge tube based on the volume.

4.

Add an equal volume of growth medium to the tube. Pipet to mix.

5.

Perform a cell count and determine cell viability. Record the number of viable cells.
# viable cells: ___________________________

6.

Transfer the desired number of cells for each ChIP reaction into separate 1.7 ml siliconized
microcentrifuge tubes (provided in the Low Cell ChIP-Seq Kit). The use of siliconized tubes
is recommended to avoid sample loss due that can result from material sticking to theplastic walls of microcentrifuge tubes. Below are the minimum number of cells that should
be used for each ChIP based on the abundance of the target protein.
Target protein

ExamplesMinimum Cell
NumberRobust histone

H3K4me1, H3K4me3, H3K27me3

1,000 cells

Low abundance histone

H3K27ac, H3K9ac, H3K9me3

5,000 cells

High abundance protein

CTCF, BRD4

10,000 cells

Low abundance protein

Transcription factors

50,000 cells

Note:

In working with low cell numbers, it is important to minimize the transfer of cellular material and/or pipetting whenever possible to avoid sample loss.

7.

If the total volume of the cell suspension is less than 50 µl, add ice-cold 1X PBS to adjust toa total volume of 50 µl. If the total volume of the cell suspension is larger than 50 µl, pellet
the cells by centrifugation in a microcentrifuge at 1250 x g for 5 minutes at 4°C. Carefully
remove all but 10 µl of supernatant from the tube to avoid disturbing the pellet. The pellet
may not be visible. Add 40 µl ice-cold 1X PBS to the tube for a total volume of 50 µl.

8.

Freshly prepare Complete Cell Fixation Solution as described on page 11.

9.

To fix cells, add 5 µl Complete Cell Fixation Solution to the 50 µl cell suspension. Do not pipette sample. Flick the tube gently to mix and incubate at room temperature for 10 minutes.

10. Stop the fixation reaction by adding 2.8 µl Stop Solution. Do not pipette sample. Flick the
tube gently to mix and incubate at room temperature for 5 minutes.
11.

Add 2 µl PIC and 2 µl PMSF to each tube. Then, add 138.2 µl ChIP Buffer and using the samewww.activemotif.com

11pipette tip, transfer the cells to a 2 ml V-bottom tube for sonication. Repeat for each sample.
The final cell suspension volume should be 200 µl.
12. Proceed to Section C: Chromatin Sonication.

Section B. Cell Fixation Starting with Small Tissue Biopsies
This protocol provides guidelines for working with small tissue biopsies. Optimization may berequired based on the tissue type used. Keep tissue samples on dry ice until fixed.
1.

Prepare Complete Tissue Fixation Solution as described on page 11.

2.

Record the weight of an empty 1.7 ml siliconized microcentrifuge tube (provided).
Tube weight ____________ mg

3.

Place a small tissue sample in the 1.7 ml siliconized tube and record the total weight.
Tube + tissue weight ______________mg

4.

Subtract the tube weight from the total weight to determine the weight of the tissue.
Tissue weight _____________ mg

5.

Calculate the expected cell number based on the conversion of 5,000 cells per mg of tissue.
# cells: ___________________________

6.

Add 1 ml Complete Tissue Fixation Solution to the tissue.

7.

Incubate for 15 minutes at room temperature with agitation.

8.

Add 50 µl Stop Solution. Incubate for 5 minutes at room temperature with agitation.

9.

Use your preferred method to generate a single cell suspension. Optimization may be required depending on the tissue type used. If possible, try to work within the siliconized tube
to avoid sample loss. Count cells with a hemacytometer. # viable cells ________________
Note:

In working with low cell numbers, it is important to minimize the transfer of cellular material and/or pipetting whenever possible to avoid sample loss.

10. Once cells are in a single cell suspension, centrifuge at 1,250 x g for 5 minutes at 4°C.
11.

Carefully remove and discard supernatant, taking care to avoid the cell pellet.

12. Resuspend cell pellet in 200 µl ChIP Buffer supplemented with 2 µl PIC and 2 µl PMSF. Using the same pipette tip, transfer the cells to a 2 ml V-bottom tube for sonication.
13. Proceed to Section C: Chromatin Sonication.

www.activemotif.com

12Section C. Chromatin Sonication
The section below describes the fragmentation of chromatin using sonication. Sonication results
may vary depending on cell type and sonication device being used. This protocol has been validated using Active Motif’s EpiShear™ Probe Sonicator in combination with an EpiShear™ Cooled
Sonication Platform to maintain probe height and temperature consistency between samples.
1.

Place the 2 ml microcentrifuge tube containing the cells into the tube cooler or packed ice.
Open cap and submerge the microtip into the liquid until the microtip is approximately
2 mm from the bottom of the tube. Sonicate according to optimized settings for the cell
type being used. A recommended starting range for cultured cells is: 25% amplitude, pulse
for 30 seconds on and 30 seconds off for a total sonication “on” time of 10 minutes (or 20
minutes elapsed time).

2.

Spin tubes at 4°C in a microcentrifuge at maximum speed (18,000 x g) for 2 minutes to pellet the cellular debris.

3.

Transfer ~200 µl of soluble chromatin to a new 1.7 ml siliconized microcentrifuge tube. Place
on ice and Proceed to Section D: Protein G Agarose Bead Preparation if pre-clearing of thechromatin will be performed the same day. Alternatively, chromatin can be stored at -80°C.

Section D. Protein G Agarose Bead Preparation1.

Set up two 1.5 ml microcentrifuge tubes to prepare the Protein G Agarose beads for the IP
and pre-clearing. We recommend cutting 2 mm from the end of a pipet tip prior to pipetting
Protein G agarose beads to prevent the tip from becoming clogged. If more than 12 IP reactions will be performed, we suggest setting up multiple tubes to ensure sufficient room inthe tube for the addition of all the required components. Volumes to add will be based on
the number of IP reactions performed. Include negative and positive control IPs into the calculation for the number of IP reactions. Volumes shown below include excess for pipetting.

Reagents

4 rxnsProtein G Agarose

140 µlIP Reactions
8 rxns 12 rxns
270 µl400 µlPre-clearing Reactions
4 rxns 8 rxns 12 rxns
140 µl270 µl400 µl* We do not recommend more than 12 IP reactions in a single tube. If more than 12 IPs are to be
performed in a single experiment, set up multiple tubes.

2.

Centrifuge the tubes at 1250 x g for 3 minutes. Carefully remove and discard supernatant,taking care to avoid the beads.

3.

Add the calculated volume of TE, pH 8.0 to the each tube of beads:
Reagents

4 rxnsIP Reactions
8 rxns 12 rxns

TE, pH 8.0

460 µl900 µl 1330 µl

www.activemotif.com

13

Pre-clearing Reactions
4 rxns 8 rxns 12 rxns
460 µl900 µl1330 µl4.

Centrifuge the tubes at 1250 x g for 3 minutes. Carefully remove and discard supernatant,taking care to avoid the beads.

5.

Set up bead blocking reactions according to the table below:
IP Reactions
8 rxns 12 rxns

Pre-clearing Reactions
4 rxns 8 rxns 12 rxns

Reagents

4 rxnsTE, pH 8.0

126 µl243 µl360 µl140 µl270 µl400 µlBlocking Reagent AM1 14 µl

27 µl

40 µl

14 µl

27 µl

40 µl

BSA

14 µl

27 µl

40 µl

14 µl

27 µl

40 µl

Blocker

14 µl

27 µl

40 µl

–

–

–

Incubate at 4°C
with rotation

Overnight

3 - 6 hours

6.

Cap tubes and incubate on an end-to-end rotator at 4°C. Follow the recommendations in
the chart above for the incubation time.

7.

Following the incubation, proceed to Section E with the pre-clearing bead reactions.

Section E. Pre-clearing the Chromatin
1.

If chromatin from Section C, Step 3 was stored at -80°C, place on ice to thaw.

2.

Remove the pre-clearing reactions from the rotator and quick spin to collect contents to thebottom of the tube. Allow the IP beads to continue to incubate on the rotator.

3.

Centrifuge the tubes at 1250 x g for 3 minutes. Carefully remove and discard supernatant,taking care to avoid the beads.

4.

Add the calculated volume of ChIP Buffer to the pre-clearing reaction. Mix by inverting.
Reagents

4 rxnsChIP Buffer–

IP Reactions
8 rxns 12 rxns––

Pre-clearing Reactions
4 rxns 8 rxns 12 rxns
460 µl900 µl1330 µl

5.

Centrifuge the tubes at 1250 x g for 3 minutes. Carefully remove and discard supernatant,taking care to avoid the beads.

6.

Perform a second wash with ChIP Buffer using the volumes below. Mix by inverting.
Reagents

4 rxnsChIP Buffer–www.activemotif.com

IP Reactions
8 rxns 12 rxns
–

–

14

Pre-clearing Reactions
4 rxns 8 rxns 12 rxns
160 µl

315 µl465 µl7.

Cut off 2 mm from the end of pipet tip and transfer 50 µl of the pre-cleared Protein G
agarose bead slurry to the 1.7 ml siliconized tube containing the sonicated chromatin from
Section C, Step 3.

8.

Add 5 µl Protease Inhibitor Cocktail and 5 µl PMSF to each tube.

9.

If using less than 200 µl chromatin, adjust the final volume of the reaction to a final volumeof 260 µl using ChIP Buffer.

10. Cap tubes and incubate on an end-to-end rotator for 2-3 hours at 4°C.

Section F. Immunoprecipitation1.

Prepare separate, labeled 1.5 ml microcentrifuge tubes for each ChIP reaction, even if thesame antibody will be used for more than one sample. Prepare the antibody mixture according to the table below and place on ice.
Reagent

Antibody Mixture

Antibody

2-4 µg

1X PBSUp to 30 µl final volume2.

Centrifuge the tubes containing the pre-clearing reactions (Section E, Step 10) in a microcentrifuge at 3500 rpm for 3 minutes. Carefully transfer the supernatant, avoiding the beadpellet, to the microcentrifuge tube containing the appropriate antibody mixture.

3.

Cap tubes and incubate on an end-to-end rotator overnight at 4°C.

4.

The next day, remove the IP reactions (from Section D, Step 5) from the rotator and quick
spin to collect contents to the bottom of the tube.

5.

Add 50 µl of the IP Reaction bead slurry to each immunoprecipitation reaction. We recommend cutting off 2 mm from the end of a pipet tip to make it easier to pipet the bead slurry.

6.

Cap tubes and incubate on an end-to-end rotator for 3-4 hours at 4°C.

www.activemotif.com

15Section G. Washing & Elution of IP reactions
1.

Label a ChIP Filtration column for each ChIP reaction. Remove the tab from the bottom of
the column and place in an empty 1 ml pipet tip box as a holder.

2.

Remove the immunoprecipitation reactions from the rotator and quick spin to collect contents to the bottom of the tube.

3.

Add 600 µl ChIP Buffer to each immunoprecipitation reaction to wash any remaining beads
off the sides of the tube and transfer the entire volume (including the Protein G agarose
beads) to its labeled column. Allow flow-through to occur by gravity.

4.

During the gravity flow, transfer 100 µl Elution Buffer AM4 per ChIP reaction to a new 1.5 ml
microcentrifuge tube. Pre-warm at 37°C. (DO NOT add to columns until Step 10 below.)5.

Wash each column with 900 µl ChIP Buffer. Allow a minute for the buffer to flow throughthe column. Wash a second time with an additional 900 µl ChIP Buffer.

6.

Wash each column with 900 µl Wash Buffer AM1. Incubate for 3 minutes on the column.
Allow the buffer to flow through the column. Wash a second time with an additional 900 µl
Wash Buffer AM1.

7.

Wash each column with 900 µl LiCl Buffer. Incubate for 3 minutes on the column. Allow thebuffer to flow through the column. Wash a second time with an additional 900 µl LiCl Buffer.

8.

Wash each column with 900 µl TE Buffer pH 8.0. Allow a minute for the buffer to flow
through the column. Wash a second time with an additional 900 µl TE Buffer pH 8.0.

9.

Transfer columns to a new 1.5 ml microcentrifuge tube. Centrifuge at 1250 x g for 3 minutesat room temperature to remove residual wash buffer.

10. Label new 1.5 ml microcentrifuge tubes. Transfer the ChIP Filtration Columns to the labeledtubes. Add 50 µl pre-warmed (37°C) Elution Buffer AM4 to each column. Incubate at room
temperature for 5 minutes. Centrifuge at 1250 x g for 3 minutes at room temperature tocollect ChIP DNA.
11.

Keeping the columns in the same microcentrifuge tube, add an additional 50 µl pre-warmed(37°C) Elution Buffer AM4 to each column. Incubate at room temperature for 5 minutes.
Centrifuge at 1250 x g for 3 minutes at room temperature to collect ChIP DNA

12. Discard the ChIP Filtration columns. The flow through contains the ChIP DNA.
www.activemotif.com

16Section H. Reversal of Cross-links and DNA Purification
1.

Transfer each eluted ChIP DNA to a 250 µl PCR tube. Add 2 µl Proteinase K and 5 µl 5 M
NaCl. Vortex to mix. Heat in a thermocycler at 65°C overnight.

2.

Following reversal of cross-links, transfer DNA to a new 1.5 ml microcentrifuge tube. Add
125 µl phenol and 64 µl chloroform/isoamyl alcohol (24:1). Shake vigorously for 15 seconds
to mix (do not vortex). Incubate at room temperature for 5 minutes.

3.

Centrifuge in a microcentrifuge at maximum speed for 2 minutes. Transfer the aqueous
layer (top layer) to a new 1.5 ml microcentrifuge tube. A hazy white layer at interface may
still be present.

4.

Add 125 µl chloroform/isoamyl alcohol (24:1) to the aqueous layer in the new tube. Shakevigorously for 15 seconds to mix (do not vortex). Incubate at room temperature for 5 minutes.

5.

During the incubation, set up new 1.5 ml microcentrifuge tubes for each ChIP reaction andadd 2 µl Carrier to each tube.

6.

Centrifuge the DNA purifications in a microcentrifuge at maximum speed (> 18,000 x g) for 2
minutes. Transfer the aqueous layer (top layer) to microcentrifuge tube containing Carrier.

7.

Add 300 µl 100% ethanol to the DNA solution and briefly vortex to mix.

8.

Place samples at -80°C for 30 minutes to precipitate the DNA.

9.

Centrifuge in a microcentrifuge at maximum speed (> 18,000 x g) for 15 minutes at 4°C.
Mark the tube where you expect the pellet to form as it may not be visible.

10. Carefully remove the supernatant with a pipet. Avoid disturbing the location of the DNA pellet.
11.

Add 500 µl 70% ethanol to each tube and invert to mix.

12. Centrifuge in a microcentrifuge at maximum speed (> 18,000 x g) for 5 minutes at 4°C. Markthe tube where you expect the pellet to form as it may not be visible.
13. Remove 400 µl supernatant and centrifuge a second time at maximum speed (> 18,000 xg) for 2 minutes at 4°C. Carefully remove as much residual ethanol as possible with a P200
pipet taking care not to disturb the pellet.
14. Air-dry the pellet for 10-15 minutes (until residual ethanol has evaporated). Resuspend thepellet in 40 µl Low-EDTA TE Buffer. Proceed to NGS library preparation. The Low Cell ChIP
DNA fragments are double-stranded and should work with any commercial DNA library
preparation kit designed for sub-nanogram levels of DNA. Please contact our technical support for guidance. Alternatively, ChIP DNA may be stored at -20°C.

www.activemotif.com

17Section I. Troubleshooting Guide
Problem/question

Recommendation

At what points in the
protocol can I stop?

The protocol may be stopped and samples stored at the times and temperatures below:
1. After chromatin shearing, -80°C.
2. During overnight incubation of pre-cleared chromatin and antibody, 4°C.
3. During overnight reversal of cross-links, 65°C
4. After DNA clean up, -20°C.

What if my tissue is toolarge to fit into the 1.7
ml siliconized tube?

If tissue is larger than 1 mm x 1 mm, place tissue in a 10 cm petri dish and add 10
ml ice-cold 1X PBS. Add 1 ml Complete Fixation Solution and use a razor blade
to chop tissue into smaller 1 mm x 1 mm pieces. Transfer tissue with the entire
volume of PBS and fixation solution to a 15 ml conical tube. Incubate for 15 minutes at room temperature with agitation. Add 550 µl Stop Solution to quench
fixation. Proceed with Section B, Step 9 to prepare a single cell suspension.

What if I want to prepare a different numberof IP reactions otherthan those listed in the
manual?

The IP and Pre-Clearing reaction volumes are provided as a guideline and include excess for pipetting. If different numbers (other than 4, 8 or 12) reactions
are to be performed adjust the volume of reagents added using the ratios listed
as a guideline. Ensure there is some excess for pipetting, but do not include alarge excess or you may run short on reagents.

A precipitation isobserved during
Protein G Agarose bead
preparation

A small precipitation or cloudiness may be observed in the IP reaction tubes
following the overnight incubation. We suggest heating the samples at 37°C for
5 minutes to dissolve the precipitation and proceed with the protocol.

High background.

Chromatin not sheared enough. Shearing should produce DNA fragmentsthat are small enough to exclude background from neighboring chromosomal
sequences, but still large enough that there is a good possibility your ampliconremains intact. We recommend 200-1200 bp fragments. If the DNA fragments
are too large, the background is increased. Consider increasing the number ofpulses for sonication. Chromatin amounts will be too small to visualize on a gel
or BioAnalyzer and still have enough material for the IP. To investigate shearing
issues, you may need to prepare extra chromatin material.
Antibody issue. Too much antibody relative to the amount of chromatin in the
ChIP reaction. Excess antibody will result in more non-specific binding, which
will be detected as increased background. We recommend using 2-4 µg ofantibody per IP.

Poor or no enrichmentwith target antibody.

Insufficient cell numbers were used. Repeat chromatin preparation using alarger number of cells. Follow the recommendations for input cell numbers
based on the robustness of the target protein.
Antibody is not ChIP-Seq validated. The antibody does not efficiently recognize
fixed proteins, either because the epitope is destroyed by fixation or because
the epitope is masked by other proteins in a larger complex. Active Motifprovides a list of Low Cell ChIP-seq validated antibodies at www.activemotif.
com/chip-lowcell.
Low-affinity antibody. Efficiency of the Low Cell ChIP-Seq not only relies on theabundance of the target protein, but the quality of the ChIP antibody. Antibodies with low binding affinities may require the use of more cells. Alternatively,
use a different antibody.
Use fresh formaldehyde, buffered with 10-15% methanol for fixation solutions.

www.activemotif.com

18

Problem/question

Recommendation

Shortage of enzyme
reagents

Pipetting enzymes at -20°C instead of placing enzymes at 0-4°C before use.
Allow enzyme reagents to equilibrate to 0-4°C for 10 minutes prior to pipetting.
Place enzymes on ice prior to use, do not use a cryocooler.

Retention of liquid in
pipette tip

Viscous reagents may stick to pipette tip, especially for non-low retention
tips. Pipette up and down several times to ensure all liquid and/or beads are
released from the pipette tip

www.activemotif.com

19Technical Services
Active Motif North America
Toll free:Direct:Fax:
E-mail:Active Motif Europe
UK Free Phone:France Free Phone:
Germany Free Phone:Direct:Fax:
E-mail:877.222.9543
760.431.1263
760.431.1351
tech_service@activemotif.com
0800/169 31 47
0800/90 99 79
0800/181 99 10+32 (0)2 653 0001
+32 (0)2 653 0050
eurotech@activemotif.com

Active Motif Japan
Direct: +81 (0)3 5225 3638
Fax: +81 (0)3 5261 8733
E-mail: japantech@activemotif.com
Active Motif China
Direct: (86)-21-20926090
Cell Phone: 18521362870
E-mail: techchina@activemotif.com

Visit Active Motif online at activemotif.com

www.activemotif.com

20