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| Status |
Public on Jul 01, 2013 |
| Title |
Induction 10min Rep2 |
| Sample type |
SRA |
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| Source name |
E. coli lambda phage lysogen heatshock 10min
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| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
strain: MG1655
sample type: λcI857 lysogen of MG1655 and E. coli
infection: Bacteriophage lambda
time: 10min
treatment: heat shock
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| Treatment protocol |
Cultures were transferred to a 42 °C water bath and incubated with shaking; samples were taken at 2, 5, 10, and 20 min. Chloramphenicol was added to 0.1 mg/ml 2 minutes before harvesting the cells in order to freeze the translating ribosomes. The cell culture was mixed with an equal mass of ice in centrifuge tubes and centrifuged at 8,000 rpm (10,000 X g) for 10 min at 4°C to harvest the cells. The cell pellet was resuspended in 0.5 ml ice-cold lysis buffer (20 mM Tris-Cl, pH 7.5, 100 mM NH4Cl, 10 mM MgCl2, 0.1 mg/ml chloramphenicol), supplemented with 50 μl of lysozyme solution (10 mg/ml?; Sigma), mixed by pipetting, and frozen for 5 min in liquid nitrogen. The suspension was thawed, refrozen, thawed again, and supplemented with 15µl of 10% deoxycholate to complete the cell lysis. Cell debris was removed by centrifugation at 10,000 rpm for 10 min at 4 °C. 100 OD260 units of the ribosome preparation was digested with 20,000 gel units of micrococcal nuclease (M0247, NEB) and 20 units of DNase I (M0303S, NEB) at 25°C for 1 hour, followed by gentle layering onto a sucrose gradient consisting of 2.5 ml layers of 10%, 20%, 30%, and 40% sucrose solutions in polysome buffer (20 mM Tris-Cl, pH 7.5, 100 mM NH4Cl, 10 mM MgCl2, 2 mM 2-mercaptoethanol, 0.1 mg/ml chloramphenicol) that had equilibrated overnight at 4°C. The gradient was centrifuged in an SW41 Ti rotor at 35,000 rpm (151,000 X g) for 3 hours at 4°C and monosome fractions were collected.
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| Growth protocol |
MG1655 and MG1655 (λcI857) incubated with shaking at 32°C to an optical density at 600 nm (OD600) of 0.4 to 0.5.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from monosomes by the hot acid phenol method.
RNA dephosphorylated by T4 polynucleotide kinase (M0236, NEB) for 1 h at 37 °C. RNA was loaded onto a 15% Mini-PROTEAN® TBE-Urea Precast Gel (BIO-RAD) and run until the ~30 nt region was resolved. The region from 28nt to 42nt was excised, based on the mobility of defined RNA oligonucleotides and a 10bp DNA ladder. The gel fraction was eluted overnight in 300 mM NaOAc pH 5.5, 1 mM EDTA, and 0.1U/ul SUPERas In (Ambion #AM2694), followed by ethanol precipitation. PolyA tails were added to the purified RNA fragments by E. coli polyA polymerase (NEB) at 37 °C for 20 min in the buffer provided. The tailed RNA molecules were reverse transcribed using barcoded primers and SuperScript III (Invitrogen) to generate the first-strand cDNA. After reverse transcription RNA was removed from RNA-DNA duplexes by incubation for 15 min at 98° in 0.1M NaOH, followed by addition of an equal concentration of HCl. Reverse-transcription products were loaded onto a 10% polyacrylamide TBE-urea gel. The band of the first-strand cDNA synthesis was excised and recovered using DNA gel elution buffer (300mM NaCl, 1mM EDTA). Purified first-strand cDNA was circularized by 50U CircLigase (Epicentre). Circularized ribosomal footprint cDNA was amplified by PCR using the Phusion High-Fidelity enzyme (New England Biolabs) according to the manufacturer’s instructions. The PCR products were separated on a non-denaturing 8% polyacrylamide TBE gel. Mixed DNA samples from different barcoded samples typically were used for cluster generation. Deep sequencing was performed with the Illumina HiSEQ2000.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Ribosome-protected mRNA
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| Data processing |
Prior to alignment, polyA tails were removed from the 3' end of reads.
SOAP2 were used to map reads onto genome allowing two or less mismatches.
RPKM of each gene was calculated by summing the number of reads on the gene location
Genome_build: n/a (e. coli and phage genes)
Supplementary_files_format_and_content: rpkm
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| Submission date |
May 30, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Huifeng Jiang |
| E-mail(s) |
jiang_hf@tib.cas.cn
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| Organization name |
Tianjin Institute of Industrial Biotechnology
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| Street address |
#32XiQiDao Tianjin airport economic park
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| City |
Tianjin |
| ZIP/Postal code |
300308 |
| Country |
China |
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| Platform ID |
GPL15010 |
| Series (1) |
| GSE47509 |
High resolution view of bacteriophage lambda gene expression by ribosome profiling |
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| Relations |
| BioSample |
SAMN02183367 |
| SRA |
SRX287637 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1151296_MG1655_cI857_10min_2.txt.gz |
87.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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