E. coli strain DH10B was purchased from Novagen. E. colistrain B7A was obtained from Dr Jaquelyn Fleckenstein (Departments of Medicine and Molecular Sciences, University of Tennessee Health Science Center).
Mono nucleosides standard samples (A, G, C, T) and PT-modified dinucleotide standard samples (GPSA, GPST) were synthesized by Sangon Biotech Co. Ltd. (Shanghai).
Pipettes, tips and microcentrifuge tubes (Eppendorf)
Ethanol (Sigma-Aldrich, #32205).
Acetic acid (Sigma-Aldrich, #33209).
I2 (Sigma-Aldrich, #D7295-20X).
dNTP (Sigma-Aldrich, #D7295-20X).
Nuclease P1 (Sigma-Aldrich, #N8630).
DNA ladder (Thermo Fisher Scientific, #10787018).
DNA Gel Loading Dye (Thermo Fisher Scientific, #R0611).
FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific, #EF0654).
Buffer 2 (New England BioLabs, #B7002S).
dATP (New England BioLabs, #N0440S).
T4 DNA ligase (New England BioLabs, #M0202S).
NaCl (Sigma-Aldrich, #746398).
EDTA (Sigma-Aldrich, #E9884).
ZnCl2 (Sigma-Aldrich, #793523).
Thiourea (Sigma-Aldrich, #T8656).
Tryptone (Sigma-Aldrich, #T7293).
Yeast Extract (Sigma-Aldrich, #Y1625).
Tris (Sigma-Aldrich, #RDD008).
Bacterial DNA Kit (OMEGA, #D3350-02).
Plasmid Mini Kit (OMEGA, #D6943-02).
Bacterial DNA Kit (OMEGA, #D3350-02).
Cycle Pure Kit (OMEGA, #D6492-02).
Quant-iT™ PicoGreen™ dsDNA Assay Kit (Thermo Fisher Scientific, #P11496).
Quick BluntingTM kit (New England BioLabs, #E1201L).
NEBNext DNA Library Prep Reagent Set for Illumina (New England BioLabs, #E6000L).
Horizontal Electrophoresis Systems (BIO-RAD, #09-0298).
PCR Systems (BIO-RAD, T100TM Thermal Cycler).
HPLC Systems (Agilent, 1260 Infinity II Analytical-Scale LC Purification System).
LC-MS/MS Systems (Agilent, HPLC 1260 - QQQ 6470).
Bioanalyzer Instrument 2100 (Agilent, #G2939BA).
Heated Circulating Baths (Thermo Fisher Scientific, #TSCIR35).
NanoDrop 2000 (Thermo Fisher Scientific, #ND2000CLAPTOP).
Qubit™4 Fluorometer (Thermo Fisher Scientific, #Q33226).
UV Spectrophotometer (HACH, #DR6000).
Constant Temperature Heater (Eppendorf, #5383000078).
Sonicator (Bandelin, Sonoplus HD2070 with Sonotrode MS72).
SPRIworks Fragment Library System (Beckman).
F1: 5‘-Phos/TTTAACCGCGAATTCCAG/ddC/ -3’(DNA, HPLC-purified)
R1: 5‘-GCTGGAATTCGCGGTTAAAT-3’ (DNA, HPLC-purified)
F2: 5‘-CAAGCAGAAGACGGAATACGA-3’(DNA, PAGE-purified)
R2: 5‘-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACG
CTCTTCCGATCTGCTGGAATTCGCGGTTAAAT-3’ (DNA, PAGE-purified)
Sample preparation (using E. coli B7A as an example)
3. Aspirate and discard the media.
4. Extract gDNA using the Bacterial DNA Kit according to the manufacturer's instructions.
5. Store eluted DNA at −80 °C until ready for use in following experiments.
[Note: For optimal DNA yields, the starting culture volume should be based on culture cell density. From experience, the cell density at 600 nm (OD600) of 2.0~3.0 is recommended.]
2. Add ~1 μg gDNA with 1 × ICA Reaction Buffer to clean microcentrifuge tube. Mix well, and incubate at 65 °C for 10 min and then slow cooled (0.1 °C/s) to 4 °C using a thermal cycler.
3. Analyze reaction sample using ethidium bromide-stained 1.0% agarose gel electrophoresis in 1 × TAE buffer containing 50 μM thiourea.
1. Dilute the mono nucleosides standards (C and T) to six different concentrations (0 ng/μl, 2 ng/μl, 4 ng/μl, 10 ng/μl, 20 ng/μl, 40 ng/μl).
[Note: The total number of A is equal to the number of T, and similarly the number of C is equal to the number of G. Thus, we could choose two mono nucleosides to draw the standard curve.]
2. Analysis samples using the High Performance Liquid Chromatography (HPLC) method, and the analysis of results as follow:
With the concentration of mono nucleosides standards (ng/μl) as the abscissa (X) and the peak area values of HPLC as the ordinate (Y), the standard curve was drawn.
[Note: Always prepare a fresh set of standards and record the samples volume for every use.]
1. Dilute the Rp configuration of the PT modified dinucleotide standards to seven different concentrations (0 fmol/μl, 2 fmol/μl, 10 fmol/μl, 20 fmol/μl, 40 fmol/μl, 100 fmol/μl, 200fmol/μl).
2. Add ~100 fmol/μl the Sp configuration of PT modified dinucleotides to each standard used as internal reference.
With the concentration of Rp configuration of the PT modified dinucleotide (fmol/μl) as the abscissa (X) and the ratio of the standard peak area to internal reference peak area values measured by LC-MS/MS as the ordinate (Y), the standard curve was drawn.
[Note: Always prepare a fresh set of standards and record the samples volume for every use.]
1. Prepare the nuclease P1 Hydrolysis Buffer (0.5 mM ZnCl2, 30 mM sodium acetate, pH 5.3).
2. Add ~20 μg purified DNA, 2 U of nuclease P1 and Hydrolysis Buffer to a clean microcentrifuge tube. Mix well, and incubate at 50 °C for 2 h using a thermal cycler.
3. Then mix in 100 mM Tris-HCl, pH 8.0 and 15 U of FastAP Alkaline Phosphatase, and incubate at 37 °C for 2 h for the totally dephosphorylation.
4. Remove the enzymes by ultrafiltration at 10000 rpm for 10 min.
5. Add ~200 μl deionized water to ultrafiltration tube and repeat step 4 until all of the sample has been transferred to the collection tube.
6. Collect the filtrate in a clean microcentrifuge tube and dry the sample.
7. Resuspend and elute the sample from step 6 in 40 μL deionized water.
8. Quantitative analysis sample by LC-MS/MS [2], and the analysis of results according to standard curves as follow:
The hydrolyzed mono nucleotides were quantified by HPLC according to the standard curve of mono nucleosides. Meanwhile, the PT modified dinucleotides were quantified by LC-MS/MS according to the standard curve of dinucleotides. Thus, the number of PT modified dinucleotides in a unit length of DNA can be calculated.
1. Cleave ~20 μg purified gDNA by iodine as described above in ICA methods.
[Note: This step is critical for removal of residual iodine and salts that may interfere with follow-up experiments.]
3. Add 10 U of FastAP Alkaline Phosphatase to the sample at 37 °C for 1h for remove terminal phosphate groups.
4. Inactivate the enzyme by heating at 75 °C for 5 min and slow cooled (0.1 °C/s) to 4 °C to assure proper complementary re-annealing.
6. Blunt end of the break sites using the Quick BluntingTM Kit at room temperature for 30 min.
7. Inactivate the enzyme by heating at 75 °C for 10 min and slow cooled as before.
9. Then mix in 1x NEB Buffer 2, 0.1 mM dATP and 15 U of Klenow Fragment (3'→5' exo-), and incubate at 37 °C for 30 min for the 3’-deoxyadenylation (namely A-tailing).
10. Inactivate the enzyme by heating at 75 °C for 20 min and slow cooled as before.
11. Clean up and elute the sample as before.
12. Combine 3 μM of custom duplex tag-sequence (see section of Primers) with 3’-deoxyadenylated ends by T4 DNA ligase at 16 °C for 16 h.
13. Inactivate the enzyme by heating at 75 °C for 10 min and slow cooled as before.
14. Clean up and elute the sample as before.
[Note: The quality of the DNA samples was measured using NanoDrop 2000 and their concentration was measured using the Quant-iT™ PicoGreen™ dsDNA Assay Kit.]
16. Concentrate samples using the Freeze Dryer and elute it in 30 μl MilliQ water.
17. Enrich the iodine-cleaved DNA linked unique tag by PCR amplification for 15 cycles using F2 and R2 as the primers.
[Note: During the PCR amplification, one of the primers used matches the marked segments attached to the ends of cleavage by iodine (F2), while the other matches the standard Illumina sequencing adaptor (R2). This allows only fragments containing the iodine-cleaved ends to be amplified, which achieves the enrichment of the PT-modified molecules.]
[Note: Agilent Technologies 2100 Bioanalyser is used to confirm successful library generation and Life Technologies Qubit 3.0 Fluorometer for quantification.]
19. Analysis the ICDS sequencing data, and the details as follow:
After completing Illumina sequencing, the reads containing tag were trimmed for adaptor and tag and done quality control as follows:
(1) Clipping the adapter sequences.
(2) Removing non-A, G, C, T bases of the 5 ' end.
(3) Trimming low-quality base (less than Q20).
(4) Removing reads with more than 10% of “N” calls.
(5) Filtering small fragments with less than 25 bp after clipping the adapter sequences and quality control, and then aligned to reference genomes by Burrows-Wheeler Aligner (BWA) and the position-wise coverage values were calculated by using the custom python script. The GAAC/GTTC sites will be defined as PT modified sites if their reads above 50 and ended at this site. Meanwhile, 10 non GAAC/GTTC sites were randomly selected as control.
2. Remove residual iodine and salts using MicroSpin G-25 columns.
[Note: This step is critical for removal of residual iodine and salts that may interfere with follow-up experiments.]
3. Shear the DNA samples to fragment lengths of 150–350 bp by sonication.
[Note: To fragment the DNA samples to a size range of 150–350 bp, using a probe sonicator as before.]
4. According to instructions provided with the NEBNext DNA Library Prep Reagent Set for Illumina, the resulting fragments were end-repaired, adenylated at the 3’ ends and ligated to Illumina paired-end adaptors.
5. Amplify DNA fragments by PCR for 15 cycles using standard Illumina adapter-specific primers.
6. Sequence the libraries constructed from step 5 on the Illumina HiSeq X Ten platform.
[Note: Agilent Technologies 2100 Bioanalyser is used to confirm successful library generation and Life Technologies Qubit 3.0 Fluorometer for quantification.]
7. Analysis the PT-IC-Seq data, and the details as follow:
All sequencing reads were trimmed for adaptor and low-quality bases and aligned to reference genomes by BWA for creating SAM files. Then, SAM files were converted to BAM files that were piled up using samtools and the results were visually performed using the Integrated Genomics Viewer 2.3 software (IGV; Broad Institute, Cambridge, MA, USA). Meanwhile, the position-wise reads number obtained from fragment terminals and across the same site were calculated respectively by using the custom python script. The PT modification frequency of each GAAC/GTTC sites were calculated by the number of reads ended at this site divided by all of the number of reads ended and crossed the same sites. In order to eliminate the false-ended reads arising from random shearing the DNA, 10 non GAAC/GTTC sites used as the control and their average PT modification frequency were calculated and used them as thresholds. The modification ratios at each PT sites in the whole genome will be calculated and analyzed if the modification frequency of those sites were above the set thresholds.