Chapter 1
Plants consist of many functionally specialized cell types , each with its own unique epigenome , transcriptome , and proteome . 
Characterization of these cell type-specific properties is essential to understanding cell fate specification and the responses of individual cell types to the environment . 
In this chapter we describe an approach to map chromatin features in specific cell types of Arabidopsis thaliana using nuclei purification from individual cell types with the INTACT method ( isolation of nuclei tagged in spe-cific cell types ) followed by chromatin immunoprecipitation and high-throughput sequencing ( ChIP-seq ) . 
The INTACT system employs two transgenes to generate affinity-labeled nuclei in the cell type of interest , and these tagged nuclei can then be selectively purified from tissue homogenates . 
The primary transgene encodes the nuclear tagging fusion protein ( NTF ) , which consists of a nuclear envelope-targeting domain , the green fluorescent protein , and a biotin ligase recognition peptide , while the second transgene encodes the E. coli biotin ligase ( BirA ) , which selectively biotinylates NTF . 
Expression of NTF and BirA in a specific cell type thus yields nuclei that are coated with biotin and can be purified by virtue of their affinity for streptavidin-coated magnetic beads . 
Compared with the original INTACT nuclei purification protocol , the procedure presented here is greatly simplified and shortened . 
After nuclei purification , we provide detailed instructions for chromatin isolation , shearing , and immunoprecipitation . 
Finally , we present a low input ChIP-seq library preparation protocol based on the nano-ChIP-seq method of Adli and Bernstein , and we describe multiplex Illumina sequencing of these libraries to produce high quality , cell type-specific epigenome profiles at a relatively low cost . 
The procedures given here are optimized for Arabidopsis but should be easily adaptable to other plant species . 
1 Introduction
Plants build their bodies by drawing on pools of undifferentiated stem cells in the meristems to produce a wide array of specialized cell types , each with its own unique form and function . 
This cell fate specification requires reprogramming of the stem cell epig-enome to establish and maintain the specific transcriptional program underlying the phenotype of a given specialized cell type . 
Understanding cellular differentiation is an important goal in developmental biology , but progress has been slowed by the technical challenges associated with isolating pure populations of specific cell types from a whole organism . 
In recent years a number of techniques have been used to characterize the molecular properties of different plant cell types . 
These methods include laser capture microdissection ( LCM ) and fluorescence-activated cell sorting ( FACS ) , which use mechanical separation to isolate whole cells of the desired type , as well as affinity-based methods such as tagged ribosome affinity purification ( TRAP ) and isolation of nuclei tagged in specific cell types ( INTACT ) , which are able to purify translating ribosomes and nuclei , respectively , from the target cell type . 
Each of these methods comes with certain advantages and limitations , and all have been reviewed in the recent literature [ 1 -- 4 ] . 
In this chapter , we describe the use of the INTACT method for characterizing the chromatin landscape in specific cell types of Arabidopsis thaliana . 
The strategy behind INTACT is to affinitylabel the nuclear envelope in a desired cell type such that labeled nuclei can be affinity-purified from a tissue homogenate , in a procedure similar to performing an immunoprecipitation . 
This system requires two transgenes : ( 1 ) the tripartite nuclear tagging fusion ( NTF ) , which encodes a nuclear envelope-targeting domain , green fluorescent protein ( GFP ) , and the biotin ligase recognition peptide ( BLRP ) ; and ( 2 ) E. coli biotin ligase ( BirA ) , which biotinylates the BLRP domain of NTF . 
The NTF transgene is expressed from a cell type-specific promoter and BirA is expressed from a constitutive promoter , such that the nuclear envelope is fluorescently labeled and biotinylated only in the cell type of interest ( Fig. 1a , b ) . 
Nuclei from the desired cell type can then be specifically purified from a tissue homogenate using streptavidin-coated magnetic beads ( Fig. 1c , d ) . 
This approach was initially applied to the characterization of the epigenome and nuclear transcriptome of Arabidopsis root epidermal cell types [ 5 , 6 ] and has subsequently been used in the Arabidopsis embryo [ 7 ] , C. elegans muscle and Drosophila mesoderm [ 8 ] , Xenopus cardiac tissue [ 9 ] , as well as tomato roots [ 10 ] . 
The protocol presented here utilizes an improved version of the originally published procedure for nuclei purification using INTACT [ 6 ] . 
In the original protocol , magnetic bead-bound nuclei were captured from a liquid column as they flowed past a magnet , which required a substantial amount of time given the volume of bead -- nuclei solution and the low flow rate required to efficiently capture highly pure bead-bound nuclei . 
The updated protocol does away with this flow-based setup in favor of capturing the bead-bound nuclei directly in a tube placed in a magnet with larger surface area to accommodate the required volume of liquid . 
This alteration greatly reduces the amount of time required and further simplifies the procedure without affecting yield or purity of target nuclei . 
Following the purification of nuclei from transgenic plants carrying the NTF and BirA transgenes , we next present an optimized chromatin immunoprecipitation ( ChIP ) protocol that can be used on as few as 10,000 purified nuclei . 
Finally , we describe the preparation of ChIP-seq libraries for the Illumina sequencing platform using an adaptation of the nano-ChIP-seq method developed by Adli and Bernstein [ 11 , 12 ] . 
The procedures laid out here can theoretically be applied to epigenome profiling of any plant cell type , given the availability of an appropriate cell type-specific promoter . 
Prepare all solutions with molecular biology-grade water . 
Commercially available products should be stored and handled according to the manufacturer 's instructions . 
To prepare , dilute 10 × NPB to 1 × concentration and add spermidine , spermine , and Roche Complete protease inhibi-tors just before starting the nuclei purification procedure . 
Keep this solution on ice and use within 1 h of preparation . 
7 . 
Nuclei Purification Buffer containing 1 % formaldehyde ( NPBf ) : 20 mM MOPS ( pH 7 ) , 40 mM NaCl , 90 mM KCl , 2 mM EDTA , 0.5 mM EGTA , 0.5 mM spermidine , 0.2 mM spermine , 1 % ( v/v ) formaldehyde ( Sigma Aldrich , catalog no. 252549 ) . 
To prepare , dilute 10 × NPB to 1 × concentration and add spermidine , spermine , and formaldehyde just before starting the nuclei purification procedure . 
Keep the solution at room temperature and use within 1 h. Formaldehyde is toxic . 
Avoid inhalation and skin exposure , and dispose of the used solution according to local regulations . 
8 . 
Nuclei Purification Buffer containing 0.1 % Triton X-100 ( NPBt ) : 20 mM MOPS ( pH 7 ) , 40 mM NaCl , 90 mM KCl , 2 mM EDTA , 0.5 mM EGTA , 0.5 mM spermidine , 0.2 mM spermine , 0.1 % ( v/v ) Triton X-100 . 
To prepare , dilute 10 × NPB to 1 × concentration and add spermidine , spermine , and Triton X-100 just before starting the nuclei purification procedure . 
Keep this solution on ice and use within 1 day of preparation . 
9 . 
2 M glycine : To make 100 ml , dissolve 15.01 g Glycine in water for a final volume of 100 ml , then filter-sterilize the solution . 
Store at 4 °C for up to 3 months . 
10 . 
DAPI staining stock solution : dissolve 10 mg of DAPI powder ( Sigma Aldrich , catalog no . 
D9542 ) in 5 ml of water , filtersterilize the solution and store at 4 °C in dark . 
Before nuclei staining , make a 1:10 dilution of the stock in water , and use within several hours . 
Stock solution can be stored at 4 °C for several months . 
11 . 
DynaMag 2 magnetic rack for 1.5 ml tubes ( Life Technologies , catalog no. 12321D ) and DynaMag 15 magnetic rack for 15 ml tubes ( Life Technologies , catalog no. 12301D ) . 
12 . 
Sterile plastic 10 ml serological pipettes ( Fisher Scientific , catalog no. 13-678-12E ) . 
13 . 
Pipet-Aid ( Fisher Scientific , catalog no.13-681-161 ) , or equivalent . 
14 . 
Porcelain 50 ml mortar and pestle ( Fisher Scientific , catalog nos . 
FB-961-A and FB-961-K ) , or equivalent . 
15 . 
Liquid nitrogen . 
16 . 
70 µM nylon cell strainers ( Fisher Scientific , catalog no. 08-771-2 ) . 
17 . 
M-280 Streptavidin Dynabeads ( Life Technologies , catalog no. 11205D ) 
18 . 
Nutator platform rotator ( Fisher Scientific , catalog n 14-062 ) , or equivalent . 
19 . 
1.5 ml Eppendorf tubes ( Fisher Scientific , catalog no . 
S348903 ) . 
20 . 
15 ml Falcon tubes ( Fisher Scientific , catalog no. 05-527-90 ) . 
21 . 
Vacuum desiccator for tissue cross-linking ( Fisher Scientific , catalog no. 08-594-16A ) . 
22 . 
Hausser Bright Line hemocytometer ( Fisher Scientific , catalog no. 02-671-1 ) , or equivalent . 
23 . 
Tabletop microcentrifuge and refrigerated centrifuge with rotor for 15 ml tubes . 
24 . 
4 °C cold room . 
1 . 
Nuclei lysis buffer : 50 mM Tris -- HCl ( pH 8 ) , 10 mM EDTA ( pH 8 ) , 1 % ( w/v ) SDS , and 1 × Roche complete protease inhibitor . 
Keep at room temperature and use within 1 h of preparation . 
2 . 
ChIP dilution buffer : 1.1 % ( v/v ) Triton X-100 , 1.2 mM EDTA ( pH 8 ) , 16.7 mM Tris -- HCl ( pH 8 ) , 167 mM NaCl . 
Keep solution on ice and use within 1 day . 
3 . 
Appropriate ChIP-grade antibodies . 
4 . 
Dynabeads protein A ( Life Technologies , catalog no. 10002D ) or protein G ( Life Technologies , catalog no. 10003D ) 5 . 
Low-salt wash buffer : 20 mM Tris -- HCl ( pH 8 ) , 150 mM NaCl , 0.1 % ( w/v ) SDS , 1 % ( v/v ) Triton X-100 , and 2 mM EDTA ( pH 8 ) . 
Keep solution on ice before use . 
Solution can be stored at 4 °C for up to 1 month . 
6 . 
High-salt wash buffer : 20 mM Tris -- HCl ( pH 8 ) , 500 mM NaCl , 0.1 % ( w/v ) SDS , 1 % ( v/v ) Triton X-100 , and 2 mM EDTA ( pH 8 ) . 
Keep solution on ice before use . 
Solution can be stored at 4 °C for up to 1 month . 
7 . 
LiCl wash buffer : 10 mM Tris -- HCl ( pH 8 ) , 250 mM LiCl , 1 % ( w/v ) sodium deoxycholate , 1 % ( v/v ) NP-40 , 1 mM EDTA ( pH 8 ) . 
Keep solution on ice before use . 
Solution can be stored at 4 °C for up to 1 month . 
8 . 
TE : 10 mM Tris ( pH 8 ) , 1 mM EDTA ( pH 8 ) . 
Keep solution on ice before use . 
Solution can be stored for several months at room temperature . 
9 . 
ChIP elution buffer : 100 mM NaHCO3 , 1 % ( w/v ) SDS . 
This solution should be kept at room temperature and used within several hours of preparation . 
10 . 
5 M NaCl : To make 1 l , dissolve 292.2 g NaCl in water for a final volume of 1 l , then autoclave or filter-sterilize the solution 
11. RNase A (Ambion, catalog no. AM2270).
12 . 
Proteinase K ( New England Biolabs , catalog no . 
P8107S ) . 
13 . 
Qiagen MinElute PCR purification kit ( Qiagen , catalog no. 28006 ) . 
14 . 
PicoGreen dsDNA quantitation kit ( Life Technologies , catalog no . 
P7589 ) . 
15 . 
Fluorometer ( e.g. , BioTek Synergy HT , Life Technologies Qubit , or equivalent ) . 
16 . 
0.6 ml low retention microcentrifuge tubes ( Fisher Scientific , catalog no. 02-681-311 ) . 
17 . 
DynaMag 2 magnetic rack for 1.5 ml tubes ( Life Technologies , catalog no. 12321D ) . 
18 . 
Diagenode Bioruptor Standard Sonicator ( Diagenode Inc. ) , or equivalent . 
19 . 
Tabletop microcentrifuge . 
20 . 
4 °C cold room . 
21 . 
100 °C heat block . 
1 . 
Primer 1 : 5 ′ - GACATGTATCCGGATGTNNNNNNNNN-3 ′ . 
N represents a degenerate position with all four nucleotides at that location . 
Prepare a 4 µM solution and store at -- 20 °C . 
BciVI site is underlined . 
2 . 
Primer 2 : 5 ′ - GACATGTATCCGGATGT-3 ′ . 
Prepare a 10 µM solution and store at -- 20 °C . 
BciVI site is underlined . 
3 . 
100 mM dNTP mix ( 25 mM each nucleotide ; New England Biolabs , catalog no . 
N0446S ) . 
Prepare dilutions of both 3 mM and 10 mM final concentration and store at − 20 °C . 
4 . 
0.1 M Dithiothreitol ( DTT , Affymetrix , catalog no. 70726 ) . 
5 . 
10 mg/ml bovine serum albumin ( BSA , New England Biolabs , catalog no . 
B9001S ) . 
6 . 
Sequenase Version 2.0 DNA sequencing kit ( Affymetrix , catalog no. 70770 ) : includes Sequenase Version 2.0 DNA Polymerase ( 13 U / µl ) , 5 × Sequenase enzyme reaction buffer , and glycerol enzyme dilution buffer . 
7 . 
ExoSAP-IT reagent for PCR cleanup ( Affymetrix , catalog no. 78200 ) . 
8 . 
Phusion Hot Start Flex DNA Polymerase and reaction buffer ( New England Biolabs , catalog no . 
M0535L ) . 
9 . 
Qiagen MinElute PCR purification kit ( Qiagen , catalog no. 28006 ) . 
10 . 
Qiagen MinElute gel extraction kit ( Qiagen , catalog no. 28606 ) 
3 Methods
11 . 
PicoGreen dsDNA quantitation kit ( Life Technologies , catalog no . 
P7589 ) . 
12 . 
BciVI enzyme and CutSmart digestion buffer ( New England Biolabs , catalog no . 
R0596L ) . 
13 . 
SYBR Green I nucleic acid gel stain ( Sigma Aldrich , catalog no . 
S9430 ) . 
14 . 
Equipment for agarose gel electrophoresis and imaging of DNA . 
15 . 
Bioo Scientific NEXTflex ChIP-Seq library preparation kit ( Bioo Scientific , catalog no. 5143-01 ) . 
16 . 
Bioo Scientific NEXTflex ChIP-Seq barcode adapters ( 12 unique barcodes ; Bioo Scientific , catalog no. 514121 ) . 
17 . 
Agencourt AMPure XP beads ( Beckman Coulter , catalog no . 
A63880 ) . 
18 . 
80 % Ethanol : mix 8 ml of molecular biology-grade ethanol ( Fisher Scientific , catalog no . 
BP2818 ) with 2 ml of water . 
Prepare fresh before use . 
19 . 
Thermal cycler with heated lid and adjustable ramp rate . 
20 . 
DynaMag 2 magnetic rack for 1.5 ml tubes ( Life Technologies , catalog no. 12321D ) . 
21 . 
Tabletop microcentrifuge . 
22 . 
Agilent Bioanalyzer . 
Carry out all procedures at room temperature unless otherwise specified . 
4 . 
Draw the tissue suspension into a 10 ml serological pipette and filter through a 70 µM nylon cell strainer into a 15 ml tube on ice . 
Spin down nuclei at 1,200 × g for 10 min at 4 °C . 
Decant the supernatant carefully without disturbing the pellet of nuclei and debris . 
5 . 
Gently resuspend the pellet in 1 ml of cold NPB by pipetting up and down and transfer the crude nuclei suspension to a 1.5 ml tube . 
Keep on ice . 
6 . 
Wash the appropriate amount of Streptavidin M280 Dynabead suspension ( 25 µl for nuclei from 1 to 3 g of tissue ; see Note 1 ) with 1 ml of ice-cold NPB in a 1.5 ml tube and collect beads on the DynaMag 2 magnetic rack . 
Discard the superna-tant and resuspend beads to their original volume with NPB ( e.g. , 25 µl ) . 
7 . 
Add 25 µl of washed and resuspended beads to the 1 ml of nuclei suspension from step 5 . 
Mix well and rotate on a nutator at 4 °C for 30 min . 
Work in the 4 °C cold room for steps 8 -- 15 . 
8 . 
Transfer the 1 ml of bead -- nuclei mixture to a 15 ml tube and gently add 13 ml of ice-cold NPBt to the mixture to bring the volume to 14 ml . 
Mix gently and place on a nutator for 30 s. 9 . 
Place the 15 ml tube in the DynaMag 15 magnetic rack for 2 min to capture the nuclei -- beads on the walls of the tube . 
10 . 
Slowly and carefully remove the NPBt supernatant with a serological pipette , taking care not to disturb the beads on the walls of the tube . 
Gently resuspend the beads in 14 ml of ice-cold NPBt , mix gently , and place on a nutator for 30 s. 11 . 
Place the tube in the DynaMag 15 magnetic rack for 2 min to capture the nuclei -- beads . 
12 . 
Repeat steps 10 and 11 . 
13 . 
Slowly and carefully remove the NPBt with a serological pipette and resuspend the beads in 1 ml of ice-cold NPBt . 
Save 25 µl of this nuclei -- bead suspension and store on ice for counting of the captured nuclei on a hemocytometer . 
14 . 
Transfer the remaining nuclei -- bead suspension to an ice-cold 1.5 ml tube and capture nuclei -- beads on a DynaMag 2 magnetic rack . 
15 . 
Remove NPBt supernatant , resuspend the beads in 20 µl of NPB , keep on ice , and proceed with the chromatin immunoprecipitation procedure . 
Alternatively , nuclei -- beads can be stored at − 80 °C until further use ( see Note 2 ) . 
16 . 
To view and count nuclei under the microscope , add 1 µl of diluted DAPI solution ( 0.2 µg / µl ) to each 25 µl sample from step 13 , mix well , and place on ice for 5 min in darkness 
1 . 
Add 120 µl of nuclei lysis buffer to the purified nuclei from step 15 of Subheading 3.1 and transfer nuclei -- buffer mix to a 0.6 ml low-retention tube . 
Vortex vigorously for 2 min to lyse the nuclei . 
2 . 
Sonicate the lysed nuclei at 4 °C in a Diagenode Standard Bioruptor water bath sonicator for 40 min using the high power setting and 45 s on/15 s off sonication cycle ( see Note 4 ) . 
3 . 
After sonication , centrifuge the lysate at 18,000 × g for 2 min at 4 °C to pellet beads and debris . 
Transfer the supernatant containing the sheared chromatin to a new 1.5 ml tube . 
4 . 
Measure the volume of fragmented chromatin using a micro-pipette , and add ice-cold ChIP dilution buffer to make a tenfold dilution of the sonicated chromatin ( e.g. , the final volume of diluted chromatin should be approximately 1.4 ml ) . 
Mix gently by inverting the tube several times , and then place the tube on ice . 
5 . 
Move 10 % of the diluted chromatin sample to a new tube and store at − 80 °C as the `` input '' chromatin fraction ( see Note 5 ) . 
The remaining diluted chromatin is enough for approximately 1 -- 4 ChIP experiments ( see Note 6 ) . 
6 . 
Divide diluted chromatin into the appropriate number of 0.6 ml low retention tubes ( or a single 1.5 ml tube if using entire chromatin sample for one ChIP experiment ) . 
Add the appropriate amount of antibody to each aliquot , mix well , and rotate on a nutator platform at 4 °C for 2 -- 5 h ( see Note 7 ) . 
7 . 
To prepare magnetic beads , add the appropriate amount of protein A Dynabead suspension ( or protein G Dynabead suspension , depending on antibody isotype ) into an ice-cold 1.5 ml tube ( Use 30 µl of bead suspension per ChIP sample ) . 
Add 1 ml of ice-cold ChIP dilution buffer , and invert the tube several times to wash the beads . 
Collect the beads on a DynaMag 2 magnet rack , decant , and resuspend the beads to their original volume with ChIP dilution buffer . 
Keep the resuspended beads on ice until use . 
8 . 
Add 30 µl of the washed protein A ( or protein G ) Dynabeads from step 7 to each ChIP sample . 
Mix well and incubate on a nutator platform at 4 °C for 1 -- 2 h. Work in the 4 °C cold room for steps 9 -- 12 . 
9 . 
Collect the beads using a DynaMag 2 magnetic rack and remove the supernatant . 
10 . 
Resuspend the beads in 0.5 ml of low-salt wash buffer and incubate the beads on a nutator platform at 4 °C for 5 min 
11 . 
Repeat steps 9 and 10 using the following series of buffers : high-salt wash , LiCl wash , and TE . 
12 . 
After the TE wash , move the beads -- buffer suspension to a new , ice-cold 0.6 ml low retention tube , collect beads on the DynaMag 2 magnet rack , and remove the supernatant . 
13 . 
Add 200 µl of ChIP elution buffer to the beads , mix well , and vortex vigorously for 5 min at room temperature . 
14 . 
Collect the beads on a DynaMag 2 magnetic rack and move the supernatant containing eluted chromatin to a new 0.6 ml low-retention tube . 
Perform all subsequent steps on both this sample and the `` input '' chromatin sample from step 5 . 
15 . 
Adjust the `` input '' chromatin sample to 200 µl with ChIP dilution buffer and then add 20 µl of 5 M NaCl to the 200 µl samples of eluted chromatin and `` input '' chromatin . 
Mix well and incubate at 100 °C for 15 min to reverse the formaldehyde cross-links . 
Centrifuge briefly at 18,000 × g to collect condensation . 
16 . 
Add 1 µl of RNase A ( 1 µg ) to each sample , mix well , and incubate for 15 min at 37 °C to digest RNA . 
Centrifuge briefly to collect condensation , and then add 1 µl of Proteinase K ( 0.8 U ) . 
Mix well and incubate for 15 min at 55 °C to digest protein and antibody , and then centrifuge briefly to collect condensation . 
17 . 
Purify the ChIP DNA and input DNA using the Qiagen MinElute PCR purification kit . 
Start by adding 1 ml of buffer PB to the ~ 220 µl sample and vortex to mix . 
18 . 
Add 700 µl of this solution to a MinElute column resting in a 2 ml collection tube . 
Centrifuge at 18,000 × g for 1 min and discard the flow-through . 
19 . 
Add the remaining solution from step 17 to the same column . 
Centrifuge at 18,000 × g for 1 min and discard the flow-through . 
20 . 
Add 750 µl of buffer PE to the column . 
Centrifuge at 18,000 × g for 1 min and discard the flow-through . 
21 . 
Centrifuge at 18,000 × g for 2 min to remove any remaining buffer PE from the column . 
22 . 
Place column in a new 1.5 ml tube , add 12 µl of room temperature elution buffer EB to the center of the column membrane , and let the column stand for 1 min . 
23 . 
Centrifuge at 18,000 × g for 1 min , discard column and place the eluted DNA on ice . 
24 . 
Measure the DNA concentration using the PicoGreen DNA quantitation kit according to the manufacturer 's instructions ( see Note 8 ) 
Given the limited quantities of DNA recovered from ChIP experiments using nuclei from individual cell types , it is generally necessary to amplify the ChIP DNA prior to construction of sequencing libraries . 
The procedure presented here uses the nano-ChIP-seq method developed by Adli and Bernstein [ 11 , 12 ] . 
This method employs four initial rounds of random priming of the ChIP and input DNA using a primer with nine random bases at the 3 ′ end and a unique sequence , including a BciVI restriction site , at the 5 ′ end . 
This primer is designed to form a hairpin at the 5 ′ end at low temperatures in order to minimize primer self-annealing . 
The priming reaction is therefore carried out using Sequenase polymerase , which is active at 37 °C but is not thermostable . 
Thus , additional enzyme is added after each cycle of priming and denaturation . 
After the four cycles of random priming , a limited number of PCR cycles are carried out using a primer corresponding to the unique 5 ′ end of the primer used in the priming step , in order to amplify the DNA and add BciVI sites to each end . 
Finally , the amplified DNA is digested with BciVI to generate 3 ′ A overhangs , and this DNA is used for conventional ChIP-seq library preparation . 
For all steps in this and the subsequent section , include both ChIP DNA and `` input '' DNA samples . 
Also include a negative control reaction ( without added DNA ) to ensure that no DNA contamination is present in the reagents or environment . 
allow the cycler to proceed through Phases 3 -- 8 ( see Table 3 ) in which the temperature gradually increases from 8 to 37 °C , then holds at 37 °C for 8 min . 
6 . 
While the thermal cycler is progressing through Phases 3 -- 8 , prepare diluted Sequenase enzyme solution ( 1:4 dilution ) by mixing 0.9 µl of Sequenase dilution buffer and 0.3 µl of the Sequenase enzyme per priming reaction . 
Prepare a master mix of the diluted Sequenase sufficient for three additions of 1.2 µl to each reaction ( i.e. , 3.6 µl per reaction ) . 
Mix well by gently pipetting the entire volume up and down several times . 
Keep on ice . 
7 . 
After the thermal cycler has passed again through Phase 1 ( 98 °C ) and has been in Phase 2 ( 8 °C ) for 1 min , pause the thermal cycler , remove and briefly centrifuge the tubes to collect condensation , and place on ice . 
To each tube add 1.2 µl of diluted Sequenase enzyme ( from step 6 ) , mix well by pipetting up and down , and return the tubes to the thermal cycler , and resume the program at 8 °C for 2 min before it proceeds to Phase 3 again . 
8 . 
Repeat step 7 two more times for a total of four rounds of priming . 
9 . 
When the priming cycles are complete , remove excess primer by adding 3 µl of ExoSAP-IT reagent to each sample , and mix well by pipetting up and down . 
Incubate the reactions at 37 °C for 15 min , followed by 80 °C for 15 min to inactivate the ExoSAP-IT . 
10 . 
Dilute the reaction product from step 9 by adding 45 µl of water and mix well by vortexing . 
11 . 
For each ChIP and input sample , set up four identical parallel PCRs in 0.2 ml tubes by using 15 µl of diluted product from step 10 as a template for each reaction . 
Set up the PCR mix according to Table 4 , mix well , and perform PCR cycling as described in Table 5 ( see Note 10 ) . 
12 . 
Pool the 4 parallel PCRs for each sample into one 1.5 ml tube . 
13 . 
Purify the DNA using the Qiagen MinElute PCR purification kit . 
Start by adding 5 volumes of buffer PB ( 1 ml ) to the samples , and vortex to mix . 
14 . 
Add 700 µl of the solution from step 13 to a MinElute column resting in a 2 ml collection tube . 
Centrifuge at 18,000 × g for 1 min and discard the flow-through . 
15 . 
Add remaining solution from step 13 to the same column . 
Centrifuge at 18,000 × g for 1 min and discard the flow-through . 
16 . 
Add 750 µl of buffer PE to the column . 
Centrifuge at 18,000 × g for 1 min and discard the flow-through 
17 . 
Centrifuge at 18,000 × g for 2 min to remove any remaining buffer PE from the column . 
18 . 
Place the column in a new 1.5 ml tube . 
Add 12 µl of room temperature elution buffer EB to the center of the column membrane , let the column stand for 1 min , and centrifuge at 18,000 × g for 1 min . 
19 . 
Add an additional 12 µl of elution buffer EB to the center of the column membrane , let the column stand for 1 min , and centrifuge at 18,000 × g for 1 min . 
Discard the column and place the eluted DNA on ice . 
20 . 
Measure the DNA concentration using the PicoGreen DNA quantitation kit according to the manufacturer 's instructions , in order to ensure that amplification was successful ( see Note 11 ) 
The amplified ChIP DNA fragments now contain BciVI sites at each end and are of sufficient quantity to prepare sequencing libraries using conventional methods . 
The DNA is first digested with BciVI to generate 3 ′ A overhangs and sequencing library adapters with 5 ′ T overhangs are ligated onto the fragments . 
The ligated DNA is then size selected and amplified again prior to sequencing . 
appropriate NEXTflex ChIP-Seq barcode adapters . 
Mix well by pipetting the entire volume up and down several times and incubate at 22 °C for 15 min . 
The remaining BciVI digested DNA can be stored at − 80 °C for later use ( see Note 12 ) . 
5 . 
Clean up the ligation product by using the MinElute PCR purification kit according to steps 13 -- 18 in Subheading 3.3.1 and store the eluted DNA on ice . 
6 . 
Mix the eluted ligation products with 1.1 µl of diluted SYBR green I gel stain ( 10,000 × stock diluted 1:1,000 in water ) and 2 µl of 10 × DNA loading dye , and incubate at room temperature for 10 min in the dark . 
Separate DNA on a freshly prepared 2 % ( w/v ) agarose gel . 
Include a DNA size marker spanning at least 100 -- 1,000 bp , in 100 bp increments . 
7 . 
Visualize the separated products on a UV light box and sizeselect the adapter-ligated DNA fragments by cutting out a gel slice corresponding to fragment sizes between 250 and 600 bp . 
8 . 
Purify the DNA from the agarose gel slice using the Qiagen QIAquick gel extraction kit . 
Weigh the gel slice in a colorless tube and add 3 volumes of Buffer QG to 1 volume of gel ( e.g. , 300 µl of QG per 100 mg of gel slice ) . 
Four hundred mg of gel is the maximum amount that can be used per purification column . 
9 . 
Incubate the gel slice in buffer QG at 50 °C for 10 min , inverting the tube every 2 -- 3 min during the incubation to mix . 
10 . 
After the gel slice has dissolved completely , check that the color of the mixture is yellow , similar to Buffer QG without dissolved agarose , indicating the correct pH ( see Note 13 ) . 
11 . 
Add 1 gel volume of isopropanol to the sample ( e.g. , 100 µl of isopropanol per 100 mg of gel slice ) and mix well by vortexing . 
12 . 
Apply up to 700 µl of the sample to a MinElute spin column resting in a 2 ml collection tube , centrifuge at 18,000 × g for 1 min , discard the flow-through , and place the MinElute column back in the same collection tube . 
13 . 
If the volume of dissolved gel solution from step 11 was greater than 700 µl , add the remainder of it to the same column and repeat step 12 . 
14 . 
Add 0.5 ml of Buffer QG to the column , centrifuge at 18,000 × g for 1 min , discard the flow-through , and place the MinElute column back in the same collection tube . 
15 . 
Add 0.75 ml of Buffer PE to the column and centrifuge at 18,000 × g for 1 min . 
16 . 
Discard the flow-through , place the MinElute column back in the same collection tube , and centrifuge the column for an additional 1 min at 18,000 × g 
17 . 
Place column into a clean 1.5 ml tube . 
To elute DNA , add 30 µl of room temperature water to the center of the membrane , let the column stand for 1 min , and then centrifuge for 1 min at 18,000 × g. Discard the column and place the eluted DNA on ice . 
18 . 
Set up the library amplification mix according to Table 8 , using reagents from the Bioo Scientific NEXTflex ChIP-Seq library preparation kit . 
Mix well by gently pipetting the entire volume up and down several times . 
Perform PCR using the thermal cycling conditions indicated in Table 9 ( see Note 14 ) . 
19 . 
Perform PCR purification by using Agencourt AMPure XP magnetic beads . 
Pre-warm the beads to room temperature and gently swirl the bottle to resuspend any magnetic particles that have settled . 
20 . 
Add 90 µl of AMPure XP beads ( 1.8 × the volume of the PCR ) to the PCR product and mix thoroughly by pipetting the entire volume up and down several times . 
Let the sample incubate for 5 min at room temperature with occasional mixing . 
21 . 
Place the tube onto the DynaMag 2 magnetic rack for 2 min to remove the beads from solution 
4 Notes
22 . 
Remove the supernatant from the tube and discard . 
23 . 
With the tubes still situated on the magnetic rack , add 200 µl of freshly prepared 80 % ethanol to each tube and incubate for 30 s. Remove the ethanol from the tubes and discard . 
Repeat this step for a total of two washes . 
24 . 
Remove the tube from the magnetic rack and allow the beads to dry for about 5 min ( see Note 15 ) . 
25 . 
Add 30 µl of room temperature water to each tube , pipette the entire volume up and down ten times to resuspend the beads , and incubate for 2 min at room temperature . 
26 . 
Place the tube on the DynaMag 2 magnetic rack for 2 min to separate beads from the solution . 
Transfer the supernatant containing eluted sequencing library DNA to a new microcentrifuge tube and place on ice . 
27 . 
Quantify DNA using the PicoGreen DNA quantitation kit according to the manufacturer 's instructions , and check the library size distribution on an Agilent Bioanalyzer . 
The library should appear as a range of fragments between approximately 200 and 600 bp ( Fig. 2a ; see Note 16 ) . 
Store the sequencing libraries at − 80 °C until use . 
28 . 
The DNA is now ready for high-throughput sequencing on the Illumina platform ( see Note 17 ) . 
Figure 2b shows a genome browser shot of typical Arabidopsis ChIP-seq data from librar-ies made using the procedures presented here . 
mark and the amount of starting chromatin . 
For example , a ChIP for H3K4me3 from 25,000 Arabidopsis nuclei will yield approximately 5 -- 10 pg of DNA . 
9 . 
During amplification and library preparation , it is essential to avoid DNA contamination from the environment . 
Ensure that all work surfaces , pipettes , and reagents are free of DNA contamination . 
10 . 
The number of PCR cycles needed should be determined empirically . 
The appropriate number of cycles to be used can be estimated by performing a test amplification on a relevant amount of `` input '' DNA and following the reaction progress in a real-time PCR instrument . 
Cycling should be stopped during the exponential phase of the reaction , and as few cycles as possible should be used . 
Using 25 cycles of PCR , 10 and 100 pg of starting DNA should yield approximately 50 and 200 ng of product , respectively . 
11 . 
It is recommended to perform qPCR to test for ChIP enrichment of one or more positive ( and negative ) control genomic regions , if such regions are known , at this step before performing the library preparations . 
12 . 
Unique barcoded adapters can be used for each sample if multiple libraries will be sequenced in an individual flow cell lane . 
Thirty nanograms of DNA in the adapter ligation reaction is recommended , but as little as 10 ng can be used . 
13 . 
If the color of the dissolved gel solution is violet or orange , this means the pH is too high . 
This can be rectified by adding 10 µl of 3 M sodium acetate ( pH 5 ) , which should bring the pH down and the color back to yellow . 
14 . 
The number of PCR cycles required for library amplification should be determined empirically , as described in Note 10 . 
Consult your sequencing core facility to determine the total amount of sequencing library DNA required for each experiment . 
15 . 
A drying time of 5 min is generally sufficient to remove all traces of ethanol from the beads , but this time may vary depending on ambient temperature and humidity . 
Care must be taken not to overdry the beads ( bead pellet will appear cracked if overdried ) , as this will negatively affect DNA elution . 
16 . 
Occasionally libraries will contain significant amounts of adapter dimers ( a distinct band of approximately 125 bp ) and / or primer dimers ( a distinct band of approximately 80 bp ) , which will negatively affect sequencing results . 
These products can be easily removed before sequencing by size selection with Agencourt SPRIselect beads ( Beckman Coulter , catalog no . 
B23317 ) 
Acknowledgements