|
| Status |
Public on Jun 19, 2024 |
| Title |
RNA-seq of E. coli wt cells in early logarithmic phase 45°C -Replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
WT
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
strain: K-12
cell type: WT
genotype: F- lambda- ilvG- rfb-50 rph-1
treatment: untreated
|
| Treatment protocol |
The resuscitated E. coli culture medium was inoculated 1:100 into LB medium and cultured at 37 ° C. When the OD600 of culture medium reached 2 (1.5 h), the culture medium of normal temperature group was kept at 37℃, and the culture medium of heat treatment group was kept at 50℃ water bath for 1 min and 20 s to increase the temperature to 45℃, and then the culture medium was kept at 45℃. Before heat treatment, 10 min and 2.5 h after heat treatment, 1 mL was sampled from the normal temperature group and the heat treatment group, respectively, to prepare 3C library
|
| Growth protocol |
E. coli cells were grown at 37°C or 45°C in Lennox Broth (LB) . The cultures were grown to early logarithmic phase or stationary phase.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was harvested using Rneasy mini plus kit (Qiagen). 1.3 ug of total RNA was used for the construction of sequencing libraries.
After the RNA samples passed the detection, magnetic beads with Oligo(dT) were used to enrich eukaryotic mRNA (if prokaryotes were prokaryotes, the mRNA was enriched by removing rRNA through the kit). Then mRNA was fragmented into fragments by fragmentation buffer. A strand of cDNA was synthesized by using mRNA as template and random Hexamers with six bases. Then buffer, dNTPs (dTTP in dNTP was replaced by dUTP) and DNA Polymerase I and RNase H were added to synthesize the double-stranded cDNA. AMPure XP Beads were used to purify the double-stranded cDNA, and USER enzyme was used to degrade the second strand of cDNA containing U. The purified double-stranded cDNA was repaired at the end, A tail was added, and sequencing adaptor was connected. Then AMPure XP beads were used for fragment size selection. Finally, PCR amplification was performed and AMPure XP beads were used to purify the PCR products to obtain the final library. After the construction of the library, Qubit 2.0 was used for preliminary quantification, and then Agilent 2100 was used to detect the size of the inserted fragments of the library. After the inserted fragments met the expectations, Q-PCR method was used to accurately quantify the effective concentration of the library to ensure the quality of the library.
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| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
CLC Genomics Workbench v 11.0.1
Sequence reads were trimmed for adaptor sequence/low-quality sequence using CLC genomic benchwork (parameter- Quality limit: 0.05)
Trimmed sequence reads were mapped to GrCM38/mm10 using CLC genomic benchwork (parameters- mismath cost: 2 insertion cost: 3 deletion cost: 3 length fraction: 0.8 similarity fraction: 0.8)
Read count extraction and normalization were performed using CLC genomic benchwork
Assembly: E. coli MG1655 GenBank: U00096.2,total length : 4639675 bp
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample
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| |
|
| Submission date |
Aug 22, 2022 |
| Last update date |
Jun 19, 2024 |
| Contact name |
binguang ma |
| E-mail(s) |
mbg@mail.hzau.edu.cn
|
| Organization name |
Huazhong Agricultural University
|
| Street address |
No.1,Shizishan Street, Hongshan
|
| City |
Wuhan |
| ZIP/Postal code |
430070 |
| Country |
China |
| |
|
| Platform ID |
GPL24659 |
| Series (2) |
| GSE211824 |
Systematic Study on the Three-Dimensional Genome of Escherichia Coli and Its Thermal Adaptation Mechanism (RNA-Seq) |
| GSE211825 |
Spatial Chromosome Organization and Adaptation of Escherichia coli under Heat Stress |
|
| Relations |
| BioSample |
SAMN30445183 |
| SRA |
SRX17236980 |
