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| Status |
Public on May 19, 2019 |
| Title |
Primary SEnd-seq WT log phase rep 4 |
| Sample type |
SRA |
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| Source name |
E.coli cell
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| Organism |
Escherichia coli |
| Characteristics |
growth stage: Log phase
treatment: 37°C culture
strain: wild type
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| Treatment protocol |
Wild type E. coli, pnp knoctout, rnb knoctout and rnr knoct out strains were cultured in LB media at 37 °C, RNA samples and ChIP-seq samples were collected when the growth stage of indicated cells reached to log phase or stationary phase. rne-3071(ts) mutation strain was cultured in LB media at 28 °C to retain the RNase E activity or shifted to 44 °C in water bath for 30 minutes to deactivate RNase E. In order to inactive the rho activity, the wild type (WT) Wild type E. coli was cultured in LB media with 50 μg/ml bicyclomycin at 37 °C for 15 min at indicated growth condition.
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| Growth protocol |
Escherichia coli K-12 MG1655 were cultured in LB media (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, pH 7.4) under aerobic conditions at 37 °C. The temperature-sensitive RNase E mutant E.coli strain [rne-3071(ts)] was cultured in LB media at 28 °C for normal culture and shift to 44 °C to deactive RNase E activity.
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| Extracted molecule |
total RNA |
| Extraction protocol |
For the RNA-seq samples, the E. coli cells were quenched by adding 0.5 volume of cold Stop Buffer (5% phenol in ethanol) immediately to the culture medium before harvest and stay on the ice for 15 minutes. Cell pellets were collected by centrifugation (6,000 rpm, 5 minutes at 4 °C), thoroughly resuspended in 100 μl of lysozyme solution [2 mg/ml in TE buffer (10 mM Tris-HCl and 1 mM EDTA)], and incubated for 2 minutes. The cells were immediately lysed by adding 1 ml of TRIzol Reagent (Invitrogen, 15596) and the RNA was extracted following manufacture’s instruction. For the ChIP-seq samples, the E. coli cells were fixed with 1% formaldehyde with continued shaking at 37 °C for 10 minutes before quenching with glycine (100 mM final concentration). Cells were then lysed and DNA was sheared by sonication followed by the treatment with micrococcal nuclease and RNase A. Antibody against RNAP β was used for target DNA enrichment.
A new RNA-seq method SEnd-seq was developed and used to prepare the SEnd-seq library, standard RNA-seq library was prepared with the TruSeq Stranded mRNA Library Prep Kit (Illumina, RS-122-2101) following the manufacturer’s instructions, ChIP-seq library was prepared with NEBNext Ultra II DNA Library Prep Kit (New England BioLabs, E7645)after ChIP DNA enrichment. For SEnd_seq, the RNA was ligated to a RNA adaptor (/5Phos/rNrNrN rNrArArCrCrUrGrCrUrArUrCrArArCrUrG/3ddC/ or /5Phos/rNrNrN rNrGrCrUrUrCrCrUrGrCrUrArUrCrArArCrUrG/3ddC/), both adaptors were used if two different samples were mixed together for library preparation(Log phase samples used the first one and stationary phase samples used the second one). The ligated RNA was converted to cDNA with the same Biotinylated RT primer(5GGGCAG T/iBiodT/GATAGCAGG). For RNA-seq , rRNA was depleted with the Ribo-Zero rRNA Removal Kit. The sequencing library was prepared with the TruSeq Stranded mRNA Library Prep Kit following the manufacturer’s instructions. For ChIP-seq, the enriched ChIP DNA and input DNA sequencing libraries were prepared with NEBNext Ultra II DNA Library Prep Kit.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Primary RNA was enriched from total RNA
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| Data processing |
Library strategy: SEnd-seq
Basecalls performed using CASAVA version 1.7
Paired-end reads were merged to single end fasta reads by usin FlASh software v1.2.11
Labled transcript 5' end and 3' end were extracted from merged single end reads and processed to paired-end reads again by custom program scripts
Extracted paired-end reads were mapped to E.coli genome (NC_00913.3) by using Bowtie2 v2.3.4.1, and single end full length sequence was generated by the paried-end data by custome transcripts and Bowtie2.
rRNA transcripts were removed and TSSs, TTSs and overlap TTSs were extracted from the remaining data.
Genome_build: Escherichia coli str. K-12 substr. MG1655, NC_000913.3
Supplementary_files_format_and_content: wig files
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| Submission date |
Jan 16, 2019 |
| Last update date |
May 20, 2019 |
| Contact name |
Xiangwu Ju |
| E-mail(s) |
xju@rockefeller.edu
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| Phone |
212-327-8842
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| Organization name |
The Rockefeller University
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| Lab |
Liu Lab
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| Street address |
1230 York Avenue
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| City |
Newyork |
| State/province |
N.Y. |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platform ID |
GPL21222 |
| Series (1) |
| GSE117737 |
Full-length RNA profiling reveals pervasive bidirectional transcription terminators in bacteria |
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| Relations |
| BioSample |
SAMN10760862 |
| SRA |
SRX5256194 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3566375_primary_send_seq_WT_log_S4_negative_strand.wig.gz |
543.5 Kb |
(ftp)(http) |
WIG |
| GSM3566375_primary_send_seq_WT_log_S4_positive_strand.wig.gz |
526.7 Kb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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