Chromatin Immunoprecipitation Assays for Myc and N-Myc

Abstract

Myc and N-Myc have widespread impacts on the chromatin state within cells, both in a gene-specific and genome-wide manner. Our laboratory uses functional genomic methods including chromatin immunoprecipitation (ChIP), ChIP-chip, and, more recently, ChIP-seq to analyze the binding and genomic location of Myc. In this chapter, we describe an effective ChIP protocol using specific validated antibodies to Myc and N-Myc. We discuss the application of this protocol to several types of stem and cancer cells, with a focus on aspects of sample preparation prior to library preparation that are critical for successful Myc ChIP assays. Key variables are discussed and include the starting quantity of cells or tissue, lysis and sonication conditions, the quantity and quality of antibody used, and the identification of reliable target genes for ChIP validation.

1 Introduction

Myc and MycN (referred hitherto as “c-Myc” and “N-Myc”) are basic helix-loop-helix transcription factors that, when dimerized with the binding partner Max, bind E-box sequences in a specific chromatin context and thereby activate expression of target genes [1]. However, Myc can also repress transcription with its partner Miz-1 (ZBTB17) in both cancer cells [2] and human embryonic stem cells (hESCs) [3]. In addition, Myc’s impact on chromatin can also manifest in a much more widespread manner, with Myc binding and affecting histone acetylation and methylation across the genome [4, 5]. Both the gene-specific and genome-wide modes of action by Myc are critical to its biological functions, including its important roles in normal development, tumorigenesis, and cellular reprogramming. However, the relationship between Myc and chromatin remodeling is complex and not fully understood. Myc is known to recruit several histone-modifying enzymes including the serine/threonine kinase PIM1 which phosphorylates serine 10 of histone H3 [6] and the histone acetyltransferases GCN5 [7] and TIP60 [8]. This is a dynamic process possibly involving cross talk between Myc and histone modifications, whereby Myc recruits chromatin-modifying enzymes which lay down marks that subsequently influence transcription, but also direct the localization of Myc. For example, the trimethylation of histone H3 at lysine 4 (H3K4me3) is involved in recruiting Myc to specific locations in the genome [1], and the persistence of these transcriptionally activating H3K4me3 marks is dependent on the presence of Myc [9].

To understand the global function of Myc on chromatin, our laboratory has been using functional genomic methods for years including chromatin immunoprecipitation (ChIP) with DNA hybridization arrays (ChIP-chip). More recently, we have changed to predominantly employ ChIP coupled to high-throughput sequencing (ChIP-seq) to analyze the genomic location of c-Myc and N-Myc (Fig. 1). ChIP-seq has key advantages over ChIP-chip, including its greater dynamic range, higher resolution, and more complete genome-wide coverage. The ChIP-seq technique must generally be optimized for each cell type and each antibody used; therefore in this chapter, we will describe an effective protocol using specific validated antibodies to c-Myc and N-Myc and discuss its application to several types of stem and cancer cells. Key variables include the starting quantity of cells or tissue, sonication conditions, as well as quantity and quality of antibody used. Performing ChIP assays for transcription factors such as Myc has specific additional considerations compared to ChIP assays for histone modifications. Myc is less abundant, less tightly bound to chromatin, and has a shorter half-life in the cell compared to histones. This means that a much higher starting cell number is required, and protein breakdown during sample preparation is a major concern. Additionally, since c-Myc and N-Myc share significant sequence similarity, it is particularly important to test for antibody cross-reactivity.

Fig. 1.

Schematic diagram illustrating major steps in the Myc ChIP process

Finding reliable Myc target genes for ChIP validation is another significant factor. The binding of Myc to E-box sequences is dependent on the chromatin context, which is cell type specific. It can be difficult to determine what genes Myc may be binding in the genome before doing the sequencing, yet the ChIP must be validated before proceeding with library preparation and sequencing, necessitating the use of PCR for specific candidate targets. In this protocol, we suggest Myc target genes that, from our extensive experience, can be effective in several cell types that we have studied. Other recent protocols have discussed ChIP-seq library preparation in some detail [10]. In this protocol we will focus on the aspects of sample preparation prior to library preparation, which are the most critical steps for successful immunoprecipitation of transcription factors such as Myc.

2 Materials

Prepare all solutions with ultrapure water and molecular biology grade reagents (see Note 1).

2.1 Cross-linking

1. Formaldehyde solution: 37 % w/w (see Note 2).

2. Quenching solution: 1.25 M glycine.

3. Wash solution: Phosphate Buffered Saline (PBS), pH 7.4. Protease inhibitors are optional (see Note 3).

2.2 Chromatin Preparation

1. Protease inhibitor tablets: Roche Complete Mini, EDTA free. Use 1 tablet per 10 ml of lysis buffer, wash buffer, or IP dilution buffer (cut to needed size).

2. Cell lysis buffer: 5 mM PIPES pH 8.0 and 85 mM KCl. Before each use, add Igepal CA-630 (Sigma) to a final concentration of 1 % (10 μl/ml) and protease inhibitors. Store at room temperature.

3. Nuclear lysis buffer: 50 mM Tris-Cl, pH 8.0, 10 mM EDTA pH 8.0, 1 % (w/v) SDS. Before use, add protease inhibitors. Store at room temperature.

   (See Note 4).

2.3 Immunoprecipitation

1. IP dilution buffer: 50 mM Tris-Cl pH 7.4, 1 mM EDTA pH 8.0, 150 mM NaCl, 0.25 % (w/v) deoxycholic acid (Fisher Scientific). Before each use, add Igepal CA-630 to a final concentration of 1 % and protease inhibitors. Store at 4 °C.

2. IP wash buffer 1: same as IP dilution buffer. Protease inhibitors are optional (see Note 3). Store at 4 °C.

3. IP wash buffer 2: 100 mM Tris-Cl, pH 9.0, 500 mM lithium chloride, 1 % (w/v) deoxycholic acid. Before use, add Igepal CA-630 to a final concentration of 1 %. Protease inhibitors are optional. Store at room temperature.

4. IP wash buffer 3: 100 mM Tris-Cl, pH 9.0, 500 mM lithium chloride, 150 mM NaCl, 1 % (w/v) deoxycholic acid. Before use, add Igepal CA-630 to a final concentration of 1 %. Protease inhibitors are optional. Store at room temperature.

5. Elution buffer: 50 mM NaHCO₃, 1 % (w/v) SDS. Store at room temperature.

6. 5 M NaCl.

7. Protein G magnetic beads, ChIP grade (Cell Signaling Technology) (see Note 5).

2.4 Small Equipment and Consumables

1. Magnetic bead rack (see Note 6).

2. Type B (loose) Dounce homogenizer for cell lysis.

3. Diagenode Bioruptor UCD 200 (allows sonication of multiple samples at once) or equivalent.

4. Sterile 2.0 ml screw cap, o-ring tubes for immunoprecipitations and for heating samples in water bath to reverse cross-links.

5. Low adhesion or siliconized tubes for final storage of samples.

6. Sterile, DNAse-free pipette tips.

7. Water bath or heating block capable of reaching 67 °C.

8. NanoDrop, or equivalent, for determining chromatin and DNA concentrations.

9. Rocker or rotator: Nutator (Clay Adams) or Labquake (Barnstead Thermolyne).

2.5 DNA Purification

1. Qiagen MinElute PCR kit or equivalent (see Note 7).

2. RNase A (Roche) reconstituted to 10 mg/ml in reagent grade water.

2.6 Antibodies

1. Suggested antibodies against c-Myc and N-Myc, along with normal IgG control antibodies, are given in Table 1.

Table 1.

Antibodies tested for ChIP assays

Antibody	Species reactivity	Vendor	Catalog number	Web link	Laboratory notes
Normal mouse IgG	None	Santa Cruz	sc-2025	www.scbt.com/datasheet-2025-normal-mouse-igg.html	Negative control antibody
Normal rabbit IgG	None	Santa Cruz	sc-2027	www.scbt.com/datasheet-2027-normal-rabbit-igg.html	Negative control antibody
N-Myc (NCM II), ChIP grade	Human, mouse	Abcam	ab16898	www.abcam.com/n-Myc-antibody-NCM-II-100-ChIP-Grade-ab16898.html	Not known to cross-react with c-Myc. Also suitable for IP. Works well for mouse ChIP in our lab
N-Myc (C-19)	Human, mouse	Santa Cruz	sc-791	www.scbt.com/datasheet-791-n-myc-c-19-antibody.html	Works well for mouse ChIP in our lab
N-Myc (B8.4.B)	Human, mouse	Santa Cruz	sc-53993	www.scbt.com/datasheet-53993-n-myc-b8-4-b-antibody.html	A lab favorite for human cells. Verified in our lab to be specific for N-Myc
N-Myc (M-50)	Mouse	Santa Cruz	sc-22836	www.scbt.com/datasheet-22836-n-myc-m-50-antibody.html	Didn’t work well in our lab. Not recommended
c-Myc (3C7)	Human, mouse	Chemicon/ Millipore	CBL434	www.millipore.com/catalogue/item/cbl434	Didn’t work well in our lab. Not recommended
c-Myc (N262)	Human, mouse	Santa Cruz	sc-764	www.scbt.com/datasheet-764-c-myc-n-262-antibody.html	Antibody strongly cross reacts with N-Myc.
c-Myc [8]	Human, mouse	Abcam	ab17355	www.abcam.com/c-Myc-antibody-8-ChIP-Grade-ab17355.html	Worked fine in our lab, but ab56 works better for hESC
c-Myc (9E11), ChIP grade	Human	Abcam	ab56	www.abcam.com/c-Myc-antibody-9E11-ChIP-Grade-ab56.html	A lab favorite. Verified in our lab to be specific for c-Myc

For all antibodies, we use 3 μg per IP. Santa Cruz refers to Santa Cruz Biotechnology

2.7 Controls

1. If enough chromatin is available, include a nonspecific IgG as one of the antibodies. It helps one judge the background and quality of the ChIP, works as a contamination check (if IgG is too high, solutions or IP may be contaminated), and serves as a reasonable negative control for PCR validation in the absence of negative control primers.

2. A stringent check for DNA contamination of the PCR reaction is to run a 100 μl sample of elution buffer through the elution, cross-link reversal, PCR purification kit, and final PCR evaluation.

3. Input or total chromatin control: there are several options for taking the input control. For regular ChIP, we routinely take input from the leftover IgG supernatant after incubation overnight. Use 10 % of the volume of one IP, including dilution buffer.

4. Alternatively, take 10 % of the volume of one IP before dilution with IP dilution buffer to use as input.

5. For ChIP-seq, save a portion ≥ 500 ng of chromatin before addition of IP dilution buffer to use as input. This quantity can be determined from the NanoDrop reading taken after step 3.2, item 8.

3 Methods

3.1 Preparation of Cross-linked Cells or Tissues

1. Tissues for ChIP may be flash frozen in liquid nitrogen and stored at −80 °C until ready to cross-link. On the day of cross-linking, keep tissues on dry ice until ready. Weigh and cut portion (about 30–80 mg per IP for Myc). Quickly chop with a clean scalpel, then immediately transfer to 15 ml polypropylene tube containing 10 ml of PBS and begin cross-linking (see Note 8).

2. Cultured cells for ChIP should be in log-phase growth and not confluent. Cells may be flash frozen before or after cross-linking. Immunoprecipitation of Myc will require more starting material than for active histone marks. Plan on 3 or more 6-well plates for pluripotent stem cells, and 4 or more 15 cm plates for adherent cell lines, or roughly 5 × 10⁷ cells per IP. Some researchers use up to 1 × 10⁸ cells for transcription factor IP.

3. Cross-link cells or tissues on a rocker in 1 % final volume of freshly diluted formaldehyde at room temperature in culture medium or in PBS. Cross-link 10 min for histones and 12–13 min for transcription factors such as Myc (see Note 9).

4. Quench the reaction by adding 1.25 M glycine (10×) to a final dilution of 0.125 M. Mix on a rocker for 5 min.

5. Wash adherent cells on the plate by decanting the cross-linking solution and rinsing plates twice with cold PBS. (Keep cells at room temperature while exposed to formaldehyde, but after cross-linking, keep PBS wash solution on ice, and spin in a 4 °C centrifuge at 1,000 × g. Optionally, add protease inhibitors to the PBS used for washing.) Transfer adherent cells from the plate to a 15 ml polypropylene tube by decanting the second wash, then scraping cells into the residual PBS with a cell lifter. Add more PBS and spin down for the final wash. Do not be surprised when cross-linked cells become hard to pool. They tend to float after cross-linking and may require additional centrifugation to pellet.

6. Tissues and suspension cells can be washed directly in the tube they were cross-linked in, three times in cold PBS in a 4 °C centrifuge at 1,000 × g.

7. After the final wash, pellet cells and remove supernatant. Proceed immediately to ChIP or flash freeze cross-linked cells in liquid nitrogen and freeze at −80 °C until needed.

3.2 Cell and Nuclear Lysis and Sonication

1. Lyse cells in cell lysis buffer with protease inhibitors and Igepal on ice for 20 min, using 1 ml lysis buffer per 5 × 10⁷ cells. Reserve 500 μl of cell lysis buffer to rinse tips and tubes if low cell number is a problem. Keep chromatin on ice at all times to avoid degradation.

2. Transfer cells to a type B (loose) Dounce homogenizer, 1 ml at a time. Dounce about 20 strokes on ice for a dilute cell preparation or 20–25 strokes for tissues or cells that are in clumps. Transfer cells to a 1.5 ml tube. Reserve 1 ml of cell lysis buffer to rinse the homogenizer after homogenizing, then add the rinse to the rest of the cells. This will increase yield, especially when there is little material to spare.

3. Spin the lysed cells at 2,500 × g for 5 min at 4 °C in a precooled centrifuge to pellet the nuclei.

4. Resuspend the pellet in nuclear lysis buffer with protease inhibitors, using no more than 100 μl per IP sample. Keep in mind that the Bioruptor will not efficiently sonicate more than 300 μl per 1.5 ml tube (see Note 10).

5. Incubate on ice for 15 min. Flick regularly to mix the nuclei. Proceed to sonication.

6. Sonicate 100–300 μl samples for the appropriate predetermined amount of time (see Note 11 and Table 2) in order to reduce fragments to the 200–500 bp size range, which is critical for efficient sequencing (see Fig. 2). Leave cells on ice between rounds of sonication, and replace warm water in the sonicator with cold water between each round of sonication (or use a cooling system to maintain water temperature; see Note 12).

7. Spin chromatin for 10 min at 20,000 × g in a precooled centrifuge at 4 °C. A large pellet of debris indicates that the sonication or cell lysis was inefficient.

8. A NanoDrop can be used at this point to get a rough idea of the chromatin yield. This is especially useful for trying to equalize input between two samples: excess can be saved at −80 °C after flash freezing in liquid nitrogen.

9. Check chromatin size by boiling a 10 μl sample for 20–30 min with 40 μl elution buffer and 2 μl of 5 M NaCl. The positive ions in the salt will stabilize the negatively charged DNA backbone. Purify the DNA using a PCR purification kit and run it on a 1 % agarose gel with EtBr. This is only an estimate. Note that it is hard to reverse the cross-links completely by boiling and chromatin will run high. If you choose to do this, we suggest doing only one check, as the chromatin has to sit and wait and may be subject to degradation. Keep chromatin on ice during the chromatin check. If the chromatin check reveals significant material above 500 bp on the agarose gel (Fig. 2), perform further cycles of sonication before proceeding.

10. Transfer equally divided supernatant (approximately 100 μl per IP) to a fresh screw-capped 2.0 ml DNase-free microfuge tube.

Table 2.

Representative sonication conditions for Subheading 3.2

Cell type	Example	Starting quantity	Suggested sonication time (min)
Tissue	Mouse testis	90 mg	30–35
Suspension aggregates	Neurospheres, embryoid bodies	5×10 cm plates	30–35
Adherent colonies	hESC	5×6-well plates	25–30
Adherent monolayers	Tet21N, mouse embryonic fibroblasts	5×15 cm plate	20–25

Starting quantities of cells can be divided into multiple IPs with different antibodies after sonication (generally 30–50 mg tissue per IP or 2–3 × 10 cm plates of cells per IP). Times given are optimized for ChIP-seq, which requires sonication to a size of 200–500 bp. ChIP without sequencing (using end-point PCR or qPCR to examine specific target genes) can tolerate somewhat larger fragments, so use about one-third less time. All sonications are performed in 100–300 μl total chromatin volume in a 1.5 ml tube using a Diagenode Bioruptor set to sonicate in cycles of 30 s high, 1 min off. Sonication is performed in 15 min increments, incubating the chromatin on ice for 5 min between increments

Fig. 2.

Agarose gel electrophoresis demonstrating appropriate chromatin size for ChIP. This chromatin check is performed prior to IP (see step 3.2, item 9) and can be used to fine-tune the sonication time. The left panel shows chromatin fragments that are too large; additional sonication cycles should be performed before continuing. The right panel shows chromatin fragments of the optimal size range. An additional, more accurate chromatin size check can be performed after the IP, using leftover supernatant from the IP (see step 3.3, item 6)

3.3 Immunoprecipitation

1. Reserve at least 500 ng of each experimental chromatin sample for input. Store at 4 °C until step 3.4, item 7 (reversing the cross-links of the samples).

2. Take sonicated chromatin samples which have been divided into aliquots in the 2.0 ml screw cap tubes, and add 4 volumes of IP dilution buffer with protease inhibitors to 1 volume chromatin.

3. In general, use about 3 μg of antibody for a Myc ChIP. Santa Cruz antibodies which are stored at 4 °C will retain their potency only a year. We use a similar amount of antibody for histone ChIP assays (see Note 13).

4. Incubate overnight on a rocker in a cold room.

5. Collect protein/antibody complexes by adding 15 μl of protein G magnetic beads (see Note 14) and incubating for 2 h at 4 °C on a rocker. Spin tubes briefly to remove any liquid clinging to the inside of the lid, then precipitate the beads by placing tubes in a magnetic rack for 1 min. Carefully pipette off the supernatant. The DNA of interest is on the beads.

6. Reserve 50 μl of each supernatant to check the sonication efficiency by reversing the cross-links overnight at 67 °C, then purifying DNA and running on an agarose gel. See step 3.4, item 9. This will give a better idea of the true chromatin size than boiling.

3.4 Washing

1. Wash magnetic beads twice with IP dilution buffer (see Note 3): take tubes out of the magnetic rack and mix by pipetting. Return tubes to the rack for at least 1 min to allow beads to settle. Remove supernatant carefully with a pipette and discard. Repeat. Avoid loss of magnetic beads. Thorough washing is important to reduce background.

2. Wash magnetic beads twice with IP wash buffer 2.

3. Wash twice with the higher stringency IP wash buffer 3.

4. Elute chromatin by adding 100 μl elution buffer per ChIP sample to the magnetic beads. Shake samples on a vortexer for 30 min.

5. Place tube in a magnetic separation rack for 1 min until beads are pelleted. Transfer the supernatant to a low-retention or siliconized tube.

6. Add 5 M NaCl to yield a final concentration of 0.54 M NaCl (12 μl per 100 μl of elution buffer mix).

7. Retrieve the input sample that was stored on the previous day. Add 4 volumes of ChIP elution buffer to 1 volume input sample. Add 12 μl 5 M NaCl per 100 μl of diluted sample.

8. Incubate all samples (IPs and inputs) in a 67 °C water bath overnight to reverse formaldehyde cross-links.

9. Allow samples to cool. Add 1 μl of RNase A and incubate for 20 min at 37 °C.

10. Purify DNA with a Qiagen MinElute PCR clean up kit. Elute each sample twice: first with 10 μl, then repeat with 12 μl. About 10 μl will be used to test enrichment by qPCR, and 10–12 μl will be used for library preparation. Plan for 2 μl to be lost as hold up volume.

3.5 Assessment of Enrichment

1. If intending to perform ChIP-seq, set aside 5–10 μl of the eluted DNA product, and keep the rest for sequencing library preparation (see Note 15). Primer sets for Myc target genes and negative controls are given in Tables 3 and 4 for ChIP assays performed on mouse and human cells, respectively (see also Fig. 3).

2. Reaction setup for end-point PCR with agarose gel electrophoresis:

   • 1.0 μl DNA eluate (undiluted) or input sample (1:50 and 1:200 dilutions).

   • 2.0 μl 10× reaction buffer.

   • 1.5 μl 25 mM MgCl₂.

   • 1.5 μl 2 mM dNTPs.

   • 1.5 μl 10 μM forward and reverse primer mix.

   • 4.0 μl 5 M betaine or combinatorial enhancer solution (CES, see Note 16).

   • 0.2 μl Taq polymerase.

   • 8.3 μl ddH2O.

   • 20 μl total reaction volume.

   • Run for 35 cycles:

     1×	3 min at 94 °C
     35×	30 s at 94 °C 30 s at annealing temperature (60 °C unless otherwise noted) 30 s at 72 °C
     1×	2 min at 72 °C

     Compare enrichment of IP sample to 1:200 and 1:50 dilutions of the input sample. A good enrichment of the target will show a signal over the 1:200 dilution and ideally equal to or greater than the 1:50 dilution.

3. Reaction setup for qPCR: include a reaction for the IgG control, particularly if negative control regions are uncertain. Dilute ChIP DNA 1:5 and input DNA 1:50 (or dilute all samples to 2 ng/μl).

   • 1.0 μl diluted ChIP or input DNA.

   • 7.5 μl 2× Absolute Blue SYBR reaction mix containing Taq polymerase.

   • 2μl 3 μM forward and reverse primer mix.

   • 4.5 μl ddH₂O.

   • 15μl total reaction volume.

   • Run on a qPCR machine with the following cycling conditions:

     1×	15 min at 95 °C
     45×	30 s at 95 °C 30 s at annealing temperature (60 °C unless otherwise noted) Melt curve from 70 to 90 °C

4. Evaluate qPCR enrichment relative to the input for each primer set:

   $$\text{Sample}\text{enrichment}=2^(\text{Cp}\text{input}-\text{Cp}\text{IP}\text{sample}).$$
   Then compute the relative enrichment by dividing the enrichment for the positive regions by the enrichment for a negative control region, or divide by IgG if good negative control primers aren’t available (see Note 17):

   $$\text{Relative}\text{enrichment}=\text{Sample}\text{enrichment}(\text{positive}\text{region})/\text{Sample}\text{enrichment}(\text{negative}\text{region}),$$
   or

   $$\text{Relative}\text{enrichment}=\text{Sample}\text{enrichment}(\text{positive}\text{region})/\text{IgG}\text{enrichment}(\text{same}\text{positive}\text{region}).$$

Table 3.

Suggested primer sets for PCR validation of Myc ChIP assays in mouse cells or tissues (Subheading 3.5)

Gene	Cell type tested	Product size (bp)	Forward	Reverse	Notes
Myc/N-Myc targets
Apex1	Neurosphere	188	ACG AAC AAC CCA GAA CCA AG	CTA AGC CAG AGA CCC TCA CG	Works for qPCR. Annealing temp=57–58 °C
CCD2	Neurosphere	221	GAC CCT CCT GCA GAA ACC TT	GGG AGA GCC AAA CCT AAA CC	Not tested for qPCR. Annealing temp=55–58 °C
Slc25A19	Neurosphere	228	CAG GTC AGG GAG GAA ACT GA	GCT CCC TCT TCA TCT GCA TC	Not tested for qPCR. Annealing temp=58 °C
Negative controls
Mmaa	MEF	125	TTC AGC CCC AGT GCA AGG TAG	TTT CTT ATG CCC CAC ATC CAG AG	Works for qPCR
Ubb1	MEF	92	CCC ACA CAA AGC CCC TCA ATC	CAG CGA AAG CGA CAG GCT AAA	Works for qPCR
Yes1	MEF	119	GGC CCA AAC GTC AGC TTT CAT	CTC CCA GGA ATG GTG GCT CTC	Works for qPCR

Annealing temperature for each primer set is 60 °C unless otherwise noted

Table 4.

Suggested primer sets for PCR validation of Myc ChIP assays in human cells or tissues (Subheading 3.5)

Gene	Cell type tested	Product size (bp)	Forward	Reverse	Notes
Myc/N-Myc targets
APEX1	Tet21N, hESC, embryoid bodies	121	GGC GGG ACC TGG TGC GGG GA	ACC GCG TCA CCC ACC GAA GCA	Works for qPCR. Also positive control for H3K9ac and H3K4me3
JUB	Tet21N	180	AAG CCC ACA GAG AGA GGT GGA AG	CCA GAA GGT GGT GCC TTT TTA TTG	Works for qPCR
POU5F1	hESC	216	GTG GCA TCC GTG AGT CTT TTG AG	GGC CCC ATA ATC TAC CTG CCT TT	Works for qPCR
RPL4	Tet21N, hESC	82	GCT TCC CGG CGC GTC CTG TGC	GGT GTG GAA CTG GGA TGT GCG GCG	Works for qPCR. Also positive control for H3K9ac and H3K4me3
RPL35	Tet21N hESC, embryoid bodies	122	TGC GCG CCA GGA TGC ACG GA	AGG CGG CCG GAT CAC AGC CCT	Works for qPCR. Also positive control for H3K9ac and H3K4me3
Negative controls
NDUFA3	Tet21N	206	TCA AGA ATG CCT GGG ACA AG	ATG GGG ATA GAT AAG GGG ATG	Not tested for qPCR
ATP50	Tet21N	232	TAC TGC CGC AGA GTT TGA TCT	CCC TTC CTG GCA TCT TAG GTA	Not tested for qPCR
TSEN34	Tet21N	204	CCA AAG AGG ATG AGA CCA GTG	TGG TGG GGA GTA CAG AGA AGA	Not tested for qPCR

Annealing temperature for each primer set is 60 °C

Fig. 3.

Representative results showing end-point PCR validation of ChIP assays in human cells using APEX1 primers given in Table 4 (see step 3.5, item 2). (a) ChIP in Tet21N neuroblastoma cells. Top panel, cells that overexpress N-Myc; bottom panel, cells after 3 days of tetracycline treatment which blocks N-Myc expression. (b) ChIP for both c-Myc and N-Myc in hESCs (two 6-well plates of cells per IP). Antibodies: N-Myc (Santa Cruz, sc-53993), c-Myc (Abcam, ab56), H3K9ac (Abcam, ab12179), and H3K4me3 (Millipore, 04–745)