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| Status |
Public on Jan 13, 2021 |
| Title |
TSS-seq (+TEX) rep2 |
| Sample type |
SRA |
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| Source name |
TSS-seq (+TEX)
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| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
terminator exonuclease (tex) treatment: yes
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| Treatment protocol |
For rifampicin-treated cells, rifampicin dissolved in methanol was added to a final concentration of 150 μg/mL with subsequent stirring for 30 min. Cell growth was monitored at OD600 nm to verify the inhibitory effects of rifampicin.
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| Growth protocol |
Glycerol stocks of E. coli K12 strain MG1655 were inoculated into M9 minimal media supplemented 0.2% glucose and grown at 30 ℃ with constant agitation overnight. Cultures were diluted 1:100 into fresh minimal medium and then cultured at 30 ℃ to mid-exponential phase (OD600 nm ~ 0.6).
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| Extracted molecule |
total RNA |
| Extraction protocol |
rRNA-depeleted total RNA extracted from the cells cultured in M9 minimal media at 30 ℃ was used to perform dRNA-seq6,7. To remove RNA with a 5’-monophosphate end, the residual mRNA was treated by 1 U terminator exonuclease (Epicentre) and 10U SUPERase-In (Invitrogen) at 30°C for 60 min. The reaction was terminated by adding 1 μL of 100 mM EDTA (pH 8.0), followed by phenol-chloroform extraction and ethanol precipitation.
The 5’-triphosphate end of primary RNA was then converted to a 5’-monophosphate end with 20 U RNA 5’ polyphosphatase (Epicentre) and 10 U SUPERase-In at 37°C for 60 min, followed by phenol-chloroform extraction and ethanol precipitation. The RNA sample was incubated with 100 pmol of 5’-RNA adapter (5’-GUUCAGAGUUCUACAGUCCGACGAUC), 20 nmol ATP, 10 U SUPERase-In, and 20 U T4 RNA Ligase at 37 °C for 3 hr. The adapter-ligated mRNA was hybridized with 100 pmol of random 3’ overhanging primer (5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNN) and then converted to cDNA by 600 U SuperScript II (Invitrogen), 30 U SUPERase-In, 30 nmol dNTPs, 1 µg actinomycin D, and 100 nmol DTT at 25 °C for 10 min and 42 °C for 60 min in order. The enzymatic reaction was inactivated by incubation at 70 °C for 15 min. The residual RNAs were removed by incubation with 1 N NaOH at 65 °C for 30 min and then neutralization with 1 N HCl. A fraction of the cDNA between 100 bp and 350 bp was extracted using Pippin PrepTM (Sage science, Beverly, MA) and dissolved in 20 µL ddH2O. The cDNA was amplified using a mixture of 1 µL cDNA, 0.5 µL Phusion Polymerase (NEB), 1 µL dNTPs (10 mM each), 1 µL SYBR green (Bio-Rad), and 1 µL of P1 and P2 primer mix (25 µM each). The samples were cycled to 94 ℃ for 10 s, 57 ℃ for 20 s, and 72 ℃ for 20 s (total 13-17 cycles). The amplification was monitored and stopped at the beginning of the saturation point. A fraction of the amplified DNA between 150 bp and 400 bp was then extracted using Pippin PrepTM. A second PCR amplification was performed with 1 µL of the cDNA to a total mass up to 1 µg with 6-8 cycles. The amplified DNA libraries were then purified and subject to next-generation sequencing.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
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| Description |
Sample 2
Processed data file(s):
TSS_TEX_Profile.gff.gz
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| Data processing |
Basecalls performed using CASAVA version 1.4
All sequencing reads were mapped to E. coli MG1655 reference genome (NC_000913) using CLC Genomics Workbench5 with the length fraction of 0.9 and the similarity of 0.99.
To capture transcriptional start site (TSS), corresponding genomic position of mapped reads start position (MRSP) was counted and stored for visual inspection using in-house scripts.
gff file is generated by in-house script
Genome_build: ASM584v1
Supplementary_files_format_and_content: .gff file. We followed that gff format definition of "http://www.sanger.ac.uk/resources/software/gff/spec.html" but some columns are ignored because they do not affect the visualization. In detail, fields are: <contig id> <source but ignored> <feature> <start> <end> <MRSP height> <strand> <frame ignored> [attributes] [comments]. In this, we defined MRSP (Mapped Reads Start Position) as a number of reads that are started at the genomic position. For the reads that are mapped to reverse strand, we assigned minus value for the visualization convenience. To reduces inter-library variance, we converted the value as of 1 million reads similar to RPKM. In biological meaning, MRSP indicates cross-linked sites for the ChIP-exo and putative TSS site TSS-seq and so on.
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| Submission date |
Jan 26, 2014 |
| Last update date |
Jan 13, 2021 |
| Contact name |
Byung-Kwan Cho |
| Organization name |
KAIST
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| Lab |
Systems and Synthetic Biology Lab
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| Street address |
291 Daehak-ro
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| City |
Daejeon |
| ZIP/Postal code |
305-701 |
| Country |
South Korea |
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| Platform ID |
GPL17439 |
| Series (2) |
| GSE54406 |
In vivo organization of bacterial transcription initiation complexes at a genome-scale [TSS-seq] |
| GSE54407 |
In vivo organization of bacterial transcription initiation complexes at a genome-scale |
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| Relations |
| BioSample |
SAMN02596596 |
| SRA |
SRX450255 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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