Here, we present a protocol to achieve near base pair resolution of protein-DNA interactions. This is obtained by exonuclease treatment of DNA fragments selectively enriched by chromatin immunoprecipitation (ChIP-exo) followed by high throughput sequencing.
Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA interactions. ChIP coupled to high throughput sequencing (ChIP-seq) is commonly used to determine the genomic location of proteins that interact with chromatin. However, ChIP-seq is hampered by relatively low mapping resolution of several hundred base pairs and high background signal. The ChIP-exo method is a refined version of ChIP-seq that substantially improves upon both resolution and noise. The key distinction of the ChIP-exo methodology is the incorporation of lambda exonuclease digestion in the library preparation workflow to effectively footprint the left and right 5' DNA borders of the protein-DNA crosslink site. The ChIP-exo libraries are then subjected to high throughput sequencing. The resulting data can be leveraged to provide unique and ultra-high resolution insights into the functional organization of the genome. Here, we describe the ChIP-exo method that we have optimized and streamlined for mammalian systems and next-generation sequencing-by-synthesis platform.
Chromatin immunoprecipitation (ChIP) is a powerful method to study mechanisms of gene regulation by selectively enriching for DNA fragments that interact with a given protein in living cells. Detection methods of ChIP-enriched DNA fragments have evolved as technology improves, from detection of a single locus (standard ChIP-qPCR) to hybridization on oligonucleotide microarrays (ChIP-chip) to high-throughput sequencing (ChIP-seq) 1. Although ChIP-seq has seen widespread application, chromatin heterogeneity and nonspecific DNA interactions have hampered data quality leading to false positives and imprecise mapping. To circumvent these limitations, Dr. Frank Pugh developed the ChIP-exo method 2. The salient feature of ChIP-exo is that it incorporates a 5' to 3' exonuclease, effectively footprinting transcription factor binding locations. As a result, the ChIP-exo methodology achieves higher resolution, greater dynamic range of detection, and lower background noise.
Although ChIP-exo is more technically challenging to master than ChIP-seq, it is being widely adopted as studies aim to gain unique ultra high-resolution insights using diverse biological systems 3-8. Indeed, ChIP-exo has been successfully applied to bacteria, yeast, mouse, rat, and human cell systems. As proof of principle, ChIP-exo was originally used to identify the precise binding motif for a handful of yeast transcription factors 2. The technique was also used in yeast to study the organization of the transcription pre-initiation complex, and to decipher subnucleosomal structure of various histones 9,10. More recently, we leveraged ChIP-exo to resolve adjacent TFIIB and Pol II binding events at human promoters, and showed that widespread divergent transcription arises from distinct initiation complexes 11.
The workflow presented here is optimized and streamlined for mammalian ChIP-exo (Figure 1). First, living primary or tissue culture cells are treated with formaldehyde to preserve in vivo protein-DNA interactions through a covalent crosslink. The cells are lysed and chromatin sheared to ~ 100 – 500 base pair size fragments. ChIP then selectively enriches for DNA fragments crosslinked to the protein of interest. At this point, ChIP-seq libraries are typically prepared, which inherently limits the detection resolution to the average fragment size of a few hundred base pairs. However, ChIP-exo overcomes this limitation by trimming the left and right 5' DNA borders of the protein-DNA crosslink site with lambda exonuclease. Sequencing libraries are then constructed from exonuclease digested DNA as detailed below. The resulting nested 5' borders represent an in vivo footprint of the protein-DNA interaction (Figure 1, step 14), and are detected by high throughput sequencing. Although the ChIP-exo methodology is more involved than ChIP-seq, transitions between most steps requires simple bead washing, which minimizes sample loss and experimental variability. Importantly, since ChIP-exo is a refined version of ChIP-seq, any sample that is successful with ChIP-seq should also be successful with ChIP-exo.
The in vivo footprinting of protein-DNA interactions with ChIP-exo results in a fundamentally distinct data structure from ChIP-seq. Although common ChIP-seq callers may be applied to ChIP-exo data, to obtain the most precise peak calls we recommend bioinformatics tools specifically designed with the unique structure of ChIP-exo data in mind. These include Genetrack, GEM, MACE, Peakzilla, and ExoProfiler 12-15.
Note: Double distilled H2O or molecular grade equivalent is recommended for all buffers and reactions mixes.
Day 0: Material Preparation and Cell Harvest
1. Buffer Preparation
2. Annealing of Adapter Oligonucleotides
Note: Specific oligonucleotide sequences may be found in the Additional Information Section.
3. In Vivo Chromatin Crosslinking with Formaldehyde
NOTE: For a typical ChIP experiment, starting material should contain approximately 50 million cells.
Day 1: Cell Lysis, Sonication, and ChIP
4. Cell Lysis
NOTE: During all cell lysis sections, the samples MUST be kept on ice or at 4 °C to minimize crosslink reversal.
5. Sonication of Nuclear Lysates
NOTE: During all sonication sections, the samples MUST be kept on ice to minimize crosslink reversal. The specific model of sonicator used in association with the protocol below may be found in the Table of Materials. Details on specific sonicator usage and guidelines for other instruments may be found in the Additional Information Section.
6. Coupling Antibody to Beads
NOTE: Never vortex or freeze/thaw magnetic beads since they will shatter and increase background signal.
7. Chromatin Immunoprecipitation (ChIP)
Day 2: ChIP Washes and On-resin Enzymatic Reactions
8. ChIP Washes
NOTE: To minimize cross contamination, briefly spin tubes between each wash. For the first aspiration, change tips between each sample. During subsequent washes, the same tip can be used as long as it did not touch the beads. See Additional Information Section for directions on proper ChIP washing.
9. Polishing Reaction
10. A-tailing Reaction
11. P7 Adapter Ligation Reaction
12. Phi-29 Nick Repair Reaction
13. Kinase Reaction
14. Lambda Exonuclease Reaction
15. RecJf Nuclease Reaction
16. Elution and Crosslink Reversal
Day 3: DNA Extraction and Adapter Ligation
17. Elution and Crosslink Reversal (continued)
18. Phenol Chloroform Isoamyl Alcohol (PCIAA) Extraction
19. P7 Primer Extension Reaction
20. A-tailing Reaction
21. P5 Adapter Ligation Reaction
22. Bead Clean-up
Note: Please see Materials section for manufacturer details on bead clean up.
Day 4: PCR and Gel Analysis
23. PCR
24. Gel Preparation, DNA Excision, and Visualization
25. Gel Purification
26. DNA Quantification
The following figures illustrate representative results from the ChIP-exo protocol presented here. In contrast to traditional ChIP-seq methodologies with few enzymatic steps, ChIP-exo requires eleven sequentially dependent enzymatic reactions (Figure 1). Thus, care must be taken at each step to ensure that each reaction component is added to its respective reaction master mix. We recommend generating a formulaic spreadsheet based on the reaction Tables to automatically perform the reaction master mix calculations, printing the resulting tables, and then checking off each item after it is added to the master mix.
We employ a number of quality control measures throughout the protocol to ensure high quality sequencing results (Figure 2). Each ChIP reaction contains three basic components: 1) sonicated chromatin extract from cells of interest, 2) an antibody directed against the protein of interest, and 3) ProteinG (or ProteinA) resin to immobilize the precipitated immune complexes. Obtaining high quality sonicated chromatin extracts (Figure 2A) can be quite challenging since sonication conditions must be optimized for each cell type and sonication instrument. It is worth spending time to optimize this step because the highest quality libraries start with a sonication result that yields DNA fragments between 100 – 500 bp (Figure 2A, "+" lane). Since formaldehyde crosslinks are labile and formaldehyde itself has a limited shelf-life, we use an electrophoretic mobility shift assay (Figure 2A, "-" lane) to verify that the sonicated extracts contain intact protein-DNA crosslinks, evident as super-shift of the DNA fragments. To assess background signal we routinely perform a "mock" ChIP that omits antibody from the ChIP reaction. A high quality ChIP-exo library preparation will have very little, if any, background signal in the "mock" ChIP ("-") relative to the specific ChIP antibody, in this case directed against Pol II. As seen in Figure 2B, traditional ChIP-seq libraries have substantially more background signal than ChIP-exo. Lastly, prior to sequencing, ChIP-exo library quality is assessed on the bioanalyzer (Figure 2C – D). Analysis accurately measures the library size distribution and detects contaminating adapter dimers (denoted by arrow) that run at 125 bp. If adapter dimers are present, they will reduce the sequencing bandwidth. Therefore, we recommend an additional bead cleanup that will efficiently remove DNA fragments less than 200 bp.
ChIP-exo is a powerful functional genomic technique because it is the only method capable of spatially resolving divergent, initiating, paused, and elongating RNA polymerase II on a genome-wide scale (Figure 3) 11. Since these adjacent binding events are tens of base pairs apart, ChIP-seq is unable to distinguish these binding events with resolving power of several hundred base pairs.
Figure 1: ChIP-exo Schematic. After ChIP, the P7 adapter is ligated to the sonication borders. Lambda exonuclease then trims DNA 5' to 3' to the crosslink point, thereby footprinting the protein-DNA interaction. After elution and crosslink reversal, primer extension synthesizes duplex DNA. Lastly, ligation of the P5 adapter marks the left and right exonuclease borders and the resulting library is subjected to high-throughput sequencing. Mapping the 5' ends of the sequence tags to the reference genome demarcates the exonuclease barrier and thus the precise site of protein-DNA crosslinking. Figure modified from Rhee and Pugh 17. Please click here to view a larger version of this figure.
Figure 2: ChIP-exo Quality Controls. Panels A-C show representative results from unrelated experiments. (A) The quality of the sonicated chromatin extract (from the human HCC1806 breast cancer cell line) is assessed by agarose gel electrophoresis on extracts with (+) and without (-) the crosslinks reversed. Intact crosslinks will cause protein-DNA complexes to migrate slower. Thus, running extract without crosslinks reversed (-) allows for the quality of the formaldehyde crosslinks to be assessed, which are critical for a successful ChIP. (B) Comparison of a mock IP (-) and Pol II (+) ChIP-exo and ChIP-seq library preparations after 21 cycles of PCR amplification. After PCR, DNA fragments 200-500 bp are excised (denoted by red hashed box) and purified using a gel extraction kit. (C-D) In panel C, the top trace represents an ideal trace from a pooled library (P1), and the bottom trace shows a pooled library (P2) that contains adapter dimers. Panel D shows the DNA density plot corresponding to the P1-2 library traces from panel C (arrow denotes adapter dimer band). Please click here to view a larger version of this figure.
Figure 3: ChIP-exo Spatially Resolves Distinct Bidirectional Transcription Initiation Complexes. (A) Smoothed distribution of strand-separated ChIP-exo tag 5' ends for Pol II, TFIIB, and TBP at the human RPS12 gene in proliferating K562 cells. (B) Averaged ChIP-exo patterns around the closest RefSeq TSS. Peak-pair tags were aligned to the TSS gene-by-gene, binned in non-overlapping 10bp intervals relative to the TSS, and then the average peak-pair density value across all TFIIB-occupied (n = 6,511) genes was plotted as a percent of the total. The "spikes" of TBP and TFIIB are indiscernible (vertically offset in inset). (C) Model based on panel B data, illustrating distinct transcription initiation complexes resolved by ChIP-exo (black trace). Pol II occupied two separate resolvable locations that coincided with sites of divergent transcription initiation ("Divergent") and "Pause" sites. This clear spatial separation of Pol II complexes indicates that divergent transcripts arise from distinct initiation complexes. The vast majority Pol II crosslinked about 50 bp downstream of the TSS at the "Pause" site, where it is expected to pause after initiating transcription. Pol II was most depleted 20 – 60 bp upstream of the TSS where the pre-initiation complex ("PIC") forms, indicating that on average it likely spends less time there than at the paused sites. This suggests that in most (but not necessarily all) cases, once Pol II is recruited, it rapidly clears the promoter and assumes a paused-state approximately 30 – 50 bp downstream of the TSS, consistent with the observation that Pol II pause release is a rate-limiting step in transcription. These adjacent initiation complexes are unresolvable by ChIP-seq (illustrated by gray fill trace) since its resolution is limited to a few hundred base pairs. Figure modified from Pugh and Venters 11. Please click here to view a larger version of this figure.
Supplemental File 1: Additional Information. Please click here to download this file.
Reagent | Volume (ml) | [Final] |
1 M HEPES-KOH (pH 7.5) | 50 | 50 mM |
5 M NaCl | 28 | 140 mM |
0.5 M EDTA | 2 | 1 mM |
100% Glycerol | 100 | 10% |
10% NP40 | 50 | 0.50% |
10% Triton X100 | 25 | 0.25% |
ddH2O | Fill to 1 L |
Table 1. Recipe for Lysis Buffer 1. Filter using 0.22 μm filter. Store in 50 ml tubes at 4 ˚C. Add 100 μl CPI stock to 50 ml of buffer just prior to use.
Reagent | Volume (ml) | [Final] |
1 M Tris-HCl (pH 8) | 10 | 10 mM |
5 M NaCl | 40 | 200 mM |
0.5 M EDTA | 2 | 1 mM |
0.5 M EGTA | 1 | 0.5 mM |
ddH2O | Fill to 1 L |
Table 2. Recipe for Lysis Buffer 2. Filter using 0.22 μm filter. Store in 50 ml tubes at 4 ˚C. Add 100 μl CPI stock to 50 ml of buffer just prior to use.
Reagent | Volume (ml) | [Final] |
1 M Tris-HCl (pH 8) | 10 | 10 mM |
5 M NaCl | 20 | 100 mM |
0.5 M EDTA | 2 | 1 mM |
0.5 M EGTA | 1 | 0.5 mM |
10% Deoxycholate | 10 | 0.10% |
N-lauroylsarcosine | 5 g | 0.5% (w/v) |
ddH2O | Fill to 1 L |
Table 3. Recipe for Lysis Buffer 3. Filter using 0.22 μm filter. Store in 50 ml tubes at 4 ˚C. Add 100 μl CPI stock to 50 ml of buffer just prior to use.
Reagent | Volume (ml) | [Final] |
10x PBS | 50 | 1x |
Bovine Serum Albumin | 2.5 g | 0.50% |
ddH2O | Fill to 500 |
Table 4. Recipe for Blocking Buffer. Filter using 0.22 μm filter. Store in 50 ml tubes at 4 ˚C. Add 100 μl CPI stock to 50 ml of buffer just prior to use.
Reagent | Volume (ml) | [Final] |
1 M HEPES (pH 7.5) | 25 | 50 mM |
0.5 M EDTA (pH 8) | 1 | 1 mM |
10% Sodium Deoxycholate | 35 | 0.70% |
10% NP40 | 50 | 1% |
1 M LiCl | 250 | 500 mM |
ddH2O | Fill to 500 |
Table 5. Recipe for RIPA Buffer. Filter using 0.22 μm filter. Store in 50 ml tubes at 4 ˚C. Add 100 µl CPI stock to 50 ml of buffer just prior to use.
Reagent | Volume (ml) | [Final] |
1 M Tris-Cl (pH 7.5) | 2.5 | 50 mM |
0.5 M EDTA | 1 | 10 mM |
20% SDS | 2.5 | 1% |
ddH2O | Fill to 50 |
Table 6. Recipe for ChIP Elution Buffer. Filter using 0.22 μm filter. Store at RT.
Reagent | Volume (ml) | [Final] |
1 M Tris-Cl (pH 7.5) | 0.5 | 10 mM |
ddH2O | Fill to 50 |
Table 7. Recipe for TE Buffer. Filter using 0.22 μm filter. Store at 4 ˚C.
Volume (μl) | [Final] | |
100 μM ExA2-iX | 75 | 15 μM |
100 μM ExA2-33 | 75 | 15 μM |
1 M Tris (pH 7.5) | 50 | 100 mM |
5 M NaCl | 5 | 50 mM |
ddH2O | 295 | – |
Total volume | 500 |
Table 8. P7 Adapter Annealing mix.
Volume (μl) | [Final] | |
100 μM ExA1-58 | 75 | 15 μM |
100 μM ExA1-13 | 75 | 15 μM |
1 M Tris (pH 7.5) | 50 | 100 mM |
5 M NaCl | 5 | 50 mM |
ddH2O | 295 | – |
Total volume | 500 |
Table 9. P5 Adapter Annealing mix.
Temp (˚C) | Time |
95 | 5 min |
72 | 5 min |
65 to 60 ramp decline | 5 min |
55 to 50 ramp decline | 3 min |
45 to 40 ramp decline | 3 min |
30 | 3 min |
20 | 3 min |
10 | 3 min |
4 | Forever |
Table 10. Adapter Annealing Program.
1x (μl) | [Final] | |
ddH2O | 39.8 | |
10x Reaction Buffer 2 | 5 | 1x |
100 µM ATP | 0.5 | 1 mM |
3 mM dNTPs | 1.7 | 100 μM |
3 U/μl T4 polymerase | 1 | 3 U |
5 U/μl Klenow | 1 | 5 U |
10 U/μl T4 Polynucleotide Kinase | 1 | 10 U |
Total reaction volume | 50 |
Table 11. Polishing master mix.
1x (μl) | [Final] | |
ddH2O | 42.3 | |
10x Reaction Buffer 2 | 5 | 1x |
3 mM dATP | 1.7 | 100 μM |
5 U/μl Klenow 3'-5' exo minus | 1 | 5 U |
Total reaction volume | 50 |
Table 12. A-tailing master mix.
1x (μl) | [Final] | |
ddH2O | 41 | |
100 mM ATP | 0.5 | 1 mM |
10x Reaction Buffer 2 | 5 | 1x |
400 U/μl T4 DNA Ligase | 1.5 | 600 U |
Reaction mix volume | 48 | |
15 mM Index Adapter | 2 | 30 picomoles |
Total reaction volume | 50 |
Table 13. P7 Adapter Ligation master mix.
1x (μl) | [Final] | |
ddH2O | 41 | |
10x Φ-29 Buffer | 5 | 1x |
3 mM dNTPs | 2.5 | 150 μM |
10 U/μl Φ-29 polymerase | 1.5 | 15 U |
Total reaction volume | 50 |
Table 14. Φ-29 Nick Repair master mix.
1x (μl) | [Final] | |
ddH2O | 43.5 | |
100 mM ATP | 0.5 | 1 mM |
10x Reaction Buffer 2 | 5 | 1x |
10 U/μl T4 Polynucleotide Kinase | 1 | 10 U |
Total reaction volume | 50 |
Table 15. Kinase Reaction master mix.
1x (μl) | [Final] | |
ddH2O | 43 | |
10x Lambda Buffer | 5 | 1x |
5 U/μl Lambda exonuclease | 2 | 10 U |
Total reaction volume | 50 |
Table 16. Lambda Exonuclease Reaction master mix.
1x (μl) | [Final] | |
ddH2O | 44 | |
10x Reaction Buffer 2 | 5 | 1x |
30 U/μl RecJf exonuclease | 1 | 30 U |
Total reaction volume | 50 |
Table 17. RecJf Nuclease Reaction master mix.
1x (μl) | [Final] | |
ddH2O | 6.45 | |
10x Φ-29 Buffer | 2 | 1x |
3 mM dNTP | 1.3 | 200 μM |
20 μM P7 Primer | 0.25 | 0.25 μM |
Reaction mix volume | 10 | |
EtOH precipitated sample | 10 | |
Total reaction volume | 20 |
Table 18. P7 Primer Extension Reaction master mix.
Temp (˚C) | Time |
95 | 5 min |
65 | 5 min |
30 | 2 min |
30 | Hold until Φ-29 is added |
30 | 20 min |
65 | 10 min |
4 | Forever |
Table 19. P7 Primer Extension program.
1x (μl) | [Final] | |
ddH2O | 5 | |
10x Reaction Buffer 2 | 3 | 1x |
3 mM dATP | 1 | 0.1 mM |
5 U/μl Klenow 3’ to 5’ exo minus | 1 | 5 U |
Reaction mix volume | 10 | |
Primer extended sample | 20 | |
Total reaction volume | 30 |
Table 20. A-tailing master mix.
1x (μl) | [Final] | |
ddH2O | 11.5 | |
10x T4 Ligase Buffer | 5 | 1x |
15 μM ExA1-58/13 adapter | 2 | 30 picomoles |
400 U/μl T4 DNA Ligase | 1.5 | 600 U |
Reaction mix volume | 20 | |
A-tailed sample | 30 | |
Total reaction volume | 50 |
Table 21. P5 Adapter Ligation master mix.
1x (μl) | [Final] | |
5x PCR Buffer | 10 | 1x (2 mM MgCl2) |
10 mM of each dNTP | 1 | 200 μM each |
20 μM P1.3 Primer | 1.25 | 0.5 μM |
20 μM P2.1 Primer | 1.25 | 0.5 μM |
2 U/μl Hot Start polymerase | 0.5 | 1 U |
Reaction mix volume | 14 | |
Bead eluted sample | 36 | |
Total reaction volume | 50 |
Table 22. PCR.
Temp (˚C) | Time | Cycles |
98 | 30 sec | 1 |
98 | 10 sec | 15-21 (depending on ChIP efficiency) |
52 | 30 sec | |
72 | 20 sec | |
72 | 2 min | 1 |
4 | Forever | Hold |
Table 23. PCR program.
Per DNA Standard (μl) | Per Sample (μl) | |
1:200 diluted buffer/dye mix | 190 | 198 |
DNA standards | 10 | |
ChIP-exo library | 2 | |
Total volume | 200 | 200 |
Table 24. Quantification.
We present a functional genomic protocol to determine the precise binding location for chromatin interacting proteins in an unbiased, genome-wide manner at near base pair resolution. The most critical step to achieve near base pair mapping resolution is the exonuclease treatment of the ChIP-enriched DNA while the immunoprecipitate remains on the magnetic resin. Ostensibly, protein complexes could potentially block in vivo footprinting of any given subunit (e.g., chromatin remodeling complexes or the nucleosome core particle). However, as reported previously 10, since formaldehyde is an inefficient crosslinker, it becomes increasingly unlikely that multiple subunits of a complex would crosslink to DNA and each other in the same cell at the same locus. Thus, in vivo footprinting of individual subunits of a protein complex, such as individual histone subunits of a nucleosome, is possible with ChIP-exo.
The most notable advantages of ChIP-exo are its near base pair resolution and low background. The ultra-high resolution permits detailed structural and spatial insights to be made on a genome-wide scale that are not currently possible with any other method. On the other hand, the primary limitation of ChIP-exo is that it is a technically challenging molecular biology methodology to master. In addition, the general limitations of the ChIP step also apply to ChIP-exo (e.g., commercial antibody availability and specificity, epitope accessibility, and relatively large number of cells required). Common pitfalls include poor quality sonicated extracts, using a non-ChIP grade antibody, and not keeping samples on ice as much as possible. Thus, sonication conditions must be carefully optimized and each antibody validated as previously described 18 to avoid the detrimental effects these parameters can have on the experimental outcome.
As far as sequencing depth for a transcription factor target, we typically aim for about 20 million uniquely aligned reads. Since Pol II and histone modifications are more broadly distributed, we aim for 30 to 50 million reads. It is important to note that since ChIP-exo has substantially less background than ChIP-seq, fewer reads are required to achieve similar sequencing depth.
The ChIP-exo technology is now being widely adopted despite its technical challenges, as robust variations on the original protocol continue to be published 17,19,20. In particular, one variation that may prove useful for difficult to ChIP proteins is called ChIP-nexus, which uses a single ligation step to increase the efficiently of the library preparation 20. In summary, ChIP-exo is an increasingly employed and powerful methodology for ultra-high resolution mapping of chromatin interacting proteins on a global scale. As the list of commercially available ChIP-grade antibodies continues to grow, future applications of the ChIP-exo methodology will be directed at mapping uncharted gene regulatory networks to understand the molecular circuitry of the cell in ultra-high resolution. In addition, ChIP-exo will likely be further refined and adapted to footprint in vivo protein-RNA interactions at near base pair resolution.
The authors have nothing to disclose.
We thank the Venters Lab and members of the Molecular Physiology and Biophysics Department for helpful discussions. Special thanks to Frank Pugh for his guidance, mentoring, and many insightful discussions on the nuances of ChIP-exo while I was a post-doctoral fellow in his laboratory.
37% formaldehyde, methanol free, 10x10ml ampules | ThermoFisher Scientific | 28908 | Section 3 |
Complete Protease Inhibitor cocktail (CPI) | Roche Life Science | 11873580001 | Sections 4, 8 |
Bioruptor sonicator | Diagenode | UCD200 | Section 5 |
15 ml polystyrene tubes | BD Falcon | 352095 | Section 5 |
MagSepharose Protein G Xtra beads | GE Healthcare | 28-9670-66 | Section 6 |
DynaMag-1.5ml Side Magnetic Rack | Invitrogen | 12321D | Sections 6-17, 22 |
Mini-Tube Rotator | Fisher Scientific | 05-450-127 | Sections 7, 22 |
T4 DNA Polymerase | New England BioLabs | M0203 | Section 9 |
DNA Polymerase I, Klenow | New England BioLabs | M0210 | Section 9 |
10x NEBuffer 2 (10x Reaction Buffer 2) | New England BioLabs | B7002 | Sections 9-11, 13, 15, 20 |
Thermomixer C | Eppendorf | 5382 | Sections 9-16 |
ATP | Roche Life Science | 010419979001 | Sections 9, 11, 13 |
dNTPs | New England BioLabs | N0447 | Sections 9, 12, 19, 23 |
T4 Polynucleotide Kinase | New England BioLabs | M0201 | Sections 9, 13 |
dATP | New England BioLabs | N0440 | Sections 10, 20 |
Klenow 3'-5' Exo Minus | New England BioLabs | M0212 | Sections 10, 20 |
T4 DNA ligase | New England BioLabs | M0202 | Sections 11, 21 |
Φ-29 DNA Polymerase | New England BioLabs | M0269 | Sections 12, 19 |
10x Φ-29 Buffer | New England BioLabs | B0269 | Sections 12, 19 |
Lambda Exonuclease | New England BioLabs | M0262 | Section 14 |
10x Lambda Buffer | New England BioLabs | B0262 | Section 14 |
RecJf Exonuclease | New England BioLabs | M0264 | Section 15 |
Proteinase K | Roche Life Science | 03115828001 | Section 16 |
Glycogen | Roche Life Science | 010901393001 | Section 18 |
10x T4 Ligase Buffer | New England BioLabs | B0202 | Section 21 |
AMPure XP (clean up) beads | Beckman Coulter | A63881 | Section 22 |
Q5 Hot Start DNA Polymerase | New England BioLabs | M0493 | Section 23 |
5x Q5 Buffer (5x PCR Buffer) | New England BioLabs | B9027 | Section 23 |
Qubit Fluorometer | Invitrogen | Q33216 | Section 25 |
QIAquick Gel Extraction Kit | Qiagen | 28704 | Section 25 |
Optical Clear Qubit tubes | Invitrogen | Q32856 | Section 26 |
Qubit dsDNA High Sensitivity Assay kit | Invitrogen | Q32851 | Section 26 |