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| Status |
Public on Jan 26, 2023 |
| Title |
ToCSV isolate from tolerant tomato line, 35 dpi, rep3 |
| Sample type |
SRA |
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| Source name |
infected leaves
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| Organism |
Tomato curly stunt virus |
| Characteristics |
tissue: infected leaves
cell line: NIL396
treatment: 35 dpi
group: tolerant tomato line
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| Treatment protocol |
Agrobacterium tumefaciens C58C1 cultures were used for inoculation containing ToCSV infectious viral clone (Accession: OK813888.1) in YEP media supplemented with 50 μg/ml of rifampicin and 50 μg/ml of kanamycin. Additionally, Agrobacterium tumefaciens C58C1 culture harbouring an empty pCambia2300 plasmid, functioned as a negative control (mock-inoculated) containing 50 μg/ml of rifampicin when inoculated in YEP media. Both cultures were incubated at 30 ˚C for 48 hours at 200 rpm followed by co-cultivation into fresh YEP media supplemented with rifampicin (50 μg/ml) and kanamycin (50 μg/ml) until an OD600 of 0.8 was reached. Agrobacterium cultures were centrifuged at 8000 g for 5 mins followed by the removal of the supernatant. The pellet was then washed in sterile water followed by centrifugation at 8000 g for 5 min. Thereafter, the pellet was resuspended in YEP and used for inoculation of tomato seedlings. The four-week-old seedlings were wounded along the stem using a hypodermic needle and each seedling was inoculated with 100 µL of resuspended YEP media. For mock-inoculated plants, a volume of 100 µL of the A. tumefaciens strain C58C1 (pCambia2300) was inoculated in each seedling. The experiment was repeated three times independently, each consisting of four mock-inoculated plants and four ToCSV-inoculated plants.
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| Growth protocol |
Plants were grown and maintained under a controlled environment in an insect-free chamber at 28 °C with a 16/8-h (light/dark) photoperiod.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The leaf tissue was snap-frozen in liquid nitrogen and stored at -80 °C until needed. Total DNA was extracted from the harvested leaf tissue using the modified cetyltrimethylammonium bromide (CTAB) extraction method. Harvested snap-frozen leaf tissue was crushed to a fine powder using a micro pestle and resuspended in 500 µL of CTAB buffer (2% (w/v) CTAB, 5 M NaCl, 0.2% 2-mercaptoethanol, 0.5 M EDTA, 1 M Tris-HCI, pH 8.0 and 2% w/v PVP) and incubated at 65 °C for 60 min. For phase separation, 500µl of chloroform: isoamyl alcohol (24:1) was added, followed by centrifugation at 13 000 g for 10 min at room temperature. The aqueous phase was extracted to a new microfuge tube, and an equal amount of isopropanol was added to precipitate the DNA. The mixture was centrifuged at 13 000 g for 10 min, and the supernatant was removed. The pellet was washed twice in 1 ml of ice-cold 70% ethanol (v/v) followed by centrifugation at 13 000 g for 5 min. The pellet was air-dried and resuspended in TE buffer (1 M Tris, pH 8, and 0.5 M EDTA) supplemented with 200 µg/ml RNase A and stored overnight at 4°C. Total DNA was subjected to restriction digestion using the restriction endonuclease BamH1 (Thermo Scientific, Waltham, USA). The endonuclease digestion was carried out in a reaction that contained 1X FastDigest ® buffer (Thermo Scientific, Waltham, USA), 1.25 U/µL BamH1 enzyme and DNA template. The reactions were incubated at 37 °C for 16 hours and inactivated at 80 °C for 5 min. Followed by the addition of proteinase K (20mg/ml) and subsequent incubation for 15 min at room temperature and 10 min. Following the proteinase K treatment, the digested DNA was purified using the DNA clean-up (Zymo Research, Irvine, CA, USA). A total of 500 ng of purified DNA was used for bisulfite conversion, using the Zymo EZ DNA methylation Gold™ kit. Bisulfite-modified DNA was used as a PCR template using Epimark Taq polymerase (New England Biolabs). PCR was carried out in a final volume of 20 µL, containing, 1X Epimark buffer, 200 µM dNTPs, 0.2 µM primer, 0.0125 U/µL PCR of the EpiMark Hot Start Taq DNA Polymerase and 100 ng DNA template. PCR for each primer set was performed independently using the Eppendorf master cycler (Merck, US) with the following cycling conditions; initial denaturation 95 °C for 30 sec, followed by 40 cycles of denaturation 95 °C for 30 sec, annealing at specific temperatures for each primer set (see supplementary file) for 60 sec, extension 68°C for 1 min and a final extension at 68°C for 5 min. To confirm positive amplification, 5µL of the PCR amplicons were separated on a 1 % agarose gel stained with 10 µg/mL ethidium bromide and visualized under ultraviolet light, using a ChemiDoc MP imaging system (Bio-Rad, USA).
TruSeq Nano (low molecular weight/adapter ligation)
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Raw sequence reads were filtered using Trimmomatic to remove low-quality sequences and sequencing adapters.
Processed reads were mapped to the reference ToCSV-genome (OK813888.1) using Bismark v0.23.1, powered by the Bowtie2 aligner. Methylation bias was corrected by end-trimming reads (7-13 nt) as guided by M-bias plots.
Methylated cytosines (in CG, CHG and CHH sequence contexts, respectively), were called using the Bismark v0.23.1 methylation extractor.
Assembly: OK813888.1
Supplementary files format and content: Bismark methylation extractor output text in tab-delimited text format, containing the positions of cytosines in the viral genome (OK813888.1) and their methylation status (+ or -).
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| Submission date |
Jan 25, 2023 |
| Last update date |
Jan 26, 2023 |
| Contact name |
Gerrit Koorsen |
| E-mail(s) |
gkoorsen@uj.ac.za
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| Phone |
+27115592821
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| Organization name |
University of Johannesburg
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| Street address |
Corner of Kingsway and University Avenue
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| City |
Johannesburg |
| ZIP/Postal code |
2006 |
| Country |
South Africa |
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| Platform ID |
GPL33059 |
| Series (1) |
| GSE223671 |
THE IDENTIFICATION OF THE METHYLATION PATTERNS OF TOMATO CURLY STUNT VIRUS IN RESISTANT AND SUSCEPTIBLE TOMATO LINES |
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| Relations |
| BioSample |
SAMN32903996 |
| SRA |
SRX19163922 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6970366_CHG_context_T3_35dpi_filtered_1_bismark_bt2_pe.txt.gz |
166.2 Mb |
(ftp)(http) |
TXT |
| GSM6970366_CHH_context_T3_35dpi_filtered_1_bismark_bt2_pe.txt.gz |
195.9 Mb |
(ftp)(http) |
TXT |
| GSM6970366_CpG_context_T3_35dpi_filtered_1_bismark_bt2_pe.txt.gz |
152.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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