 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 21, 2020 |
| Title |
YF11[Flag-DndC]-1-ctrl |
| Sample type |
SRA |
| |
|
| Source name |
Salmonella enterica serovar Cerro 87
|
| Organism |
Salmonella enterica |
| Characteristics |
serovar: Cerro 87
treatment: none
role: negative control
genotype: {delta}dndC [pFlag-DndC]
|
| Treatment protocol |
Fragmentation of genomic DNA. To ensure a relatively random fragmentation of genomic DNA, 1 μg of the extracted DNA was digested separately by three DNA endonuclease combinations: NciI; HindIII and XhoI; SalI, XbaI and NdeI. RNase A was also added to each digestion mixture to remove any trace of contaminated RNA. After digestion, the digested products were purified by Qiagen PCR purification Kit. The three purified DNA sample were mixed together for the following blocking step.
Blocking of pre-existing strand break sites. Reaction mixture (40 μl) containing 4 μl of reaction buffer (NEBcutsmart buffer); 1 μl of Shrimp Alkaline Phosphatase (rSAP, NEB), 1 μg of template genomic DNA was incubated at 37 °C for 30 min to remove any pre-existing strand breaks with 3' end phosphate, then 70°C for 10min to inactivate rSAP. After cool down the reaction product to room temperature, add 2 μl of ddNTPs (2.5 mM each, TriLink) and 1 μl of DNA polymerase I (10 U, NEB), incubate at 37 °C for 40 min to block any pre-existing strand break sites. Then 1 μl of Shrimp Alkaline Phosphatase (rSAP, NEB) was added to the reaction mixture, incubate at 37 °C for another 30 min to degrade the excess ddNTPs. Reaction was terminated by heat-inactivation of rSAP and DNA polymerase I at 75 °C for 10 min. The reaction product was ready for one of the following nick creation or conversion procedure after de-salts by DyeEx column (QIAGEN)
|
| Growth protocol |
S. enterica 87 and E. coli K12 expressing Salmonella DndBCDE strains were grown in Luria-Bertani (LB) at 37 °C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Nick conversion of DNA PT sites by Ioline cleavage. 40 μl of the blocked DNA was added with 5 μl dibasic Sodium phosphate buffer (500mM, pH 9.0) and 2 μl of Iodine solution (0.1N, FLUKA). After incubate at 65°C for 5 minutes and cool down slowly to 4°C, the reaction product was purified by DyeEX column (QIAGEN) to remove salts and Iodine. The purified product was treated with rSAP by adding 5 μl of NEBcutsmart buffer and 1 μl of rSAP to remove any 3' end phosphate left from iodine cleavage. After incubation at 37 °C for 20 min and 75 °C for another 10 min, the product was kept on ice for the following step.
Illumina library preparation was performed by the Clontech SMART ChIp-seq kit (Clontech) by following the manufacturer’s protocol. 12 cycles were used in the final step of PCR amplification. The PCR product from each sample was combined with its corresponding negative control and then size selected using AMPure XP beads (NEB). The purified library was submitted to Illumina HiSeq PE150 instrument for 75 bp paired-end sequencing.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
HiSeq X Ten |
| |
|
| Data processing |
Library strategy: nick-seq
Sequencing results were processed on the Galaxy web platform (https://usegalaxy.org/). Initially, the paired end reads were pre-processed by trim galore to remove adapters and the first 3 bp on the 5’ of read 1 were also trimmed.
All the reads were aligned to the corresponding genome using Bowtie 2.
A custom method for peak calling of sequencing data was developed with BamTools, BEDTools and Rstudio. Briefly, the bam results were filtered based on R1 (selected for nick translation data) or R2 (selected for 3’ capture data). The 5’ coverage (experiment sample) or full coverage (controls) on each position were calculated based on the filtered bam result by BEDTools(positive and negative strand separately)
The coverage of positionN (sample)/ coverage of positionN-1(sample), coverage of positionN(sample)/ coverage of positionN+1(sample) and coverage of positionN(sample)/ coverage of positionN(control) ratio was calculated on each position and the positions with all the three ratios above 5 were regarded as strong signals.
Genome_build: CP009273.1 and CP008925.1
Supplementary_files_format_and_content: tabular files were generated by BEDTools containing the 5’ coverage (experiment sample) or full coverage (controls) on each position (positive and negative strand separately)
|
| |
|
| Submission date |
Aug 16, 2019 |
| Last update date |
May 21, 2020 |
| Contact name |
Xiaolin Wu |
| E-mail(s) |
xiaolinwu@whu.edu.cn
|
| Organization name |
Singapore-MIT Alliance for Research and Technology (SMART)
|
| Street address |
1 CREATE Way
|
| City |
Singapore |
| State/province |
Singapore |
| ZIP/Postal code |
138602 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL27121 |
| Series (2) |
| GSE135910 |
Mapping PT modifications in bacteria |
| GSE135949 |
Epigenetic competition reveals density-dependent regulation and target site plasticity of phosphorothioate epigenetics in bacteria |
|
| Relations |
| BioSample |
SAMN12588595 |
| SRA |
SRX6728049 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4037781_YF11_FlagDndC_1_ctrl_5CoverageForward.tabular.txt.gz |
11.8 Mb |
(ftp)(http) |
TXT |
| GSM4037781_YF11_FlagDndC_1_ctrl_5CoverageReverse.tabular.txt.gz |
11.8 Mb |
(ftp)(http) |
TXT |
| GSM4037781_YF11_FlagDndC_1_ctrl_FullCoverageForward.tabular.txt.gz |
12.7 Mb |
(ftp)(http) |
TXT |
| GSM4037781_YF11_FlagDndC_1_ctrl_FullCoverageReverse.tabular.txt.gz |
12.7 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
