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| Status |
Public on May 15, 2020 |
| Title |
VC_miaA_24h |
| Sample type |
SRA |
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| Source name |
VC_miaA_24h
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| Organism |
Vibrio cholerae |
| Characteristics |
strain: C6706
growth phase: stationary phase
genotype: DmiaA
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| Extracted molecule |
total RNA |
| Extraction protocol |
Bacterial cells in a culture medium or cecal fluid were harvested by centrifugation. Total RNA was immediately extracted from cell pellets with Trizol according to the manufacture’s instruction.
The tRNA fraction was cut out from 10% TBE-Urea gels and recovered by isopropanol precipitation. tRNA fraction (250 ng) was deacylated in 500 ul of 100 mM Tris-HCl pH 9.0 at 37 °C for 1 hr and recovered by isopropanol precipitation. After dephosphorylation with alkaline phosphatase from calf intestine (New England Biolabs), tRNAs were ligated to 100 pmol of 5’ adenylated and 3’ end-blocked DNA oligo (3’ linker) using truncated T4 RNA ligase at 25 °C for 2.5 hr in 25% PEG 8000. The ligated product was purified on a 10 % TBE-Urea polyacrylamide gel (Thermo Fisher Scientific) as above. Half of the recovered ligated tRNAs were reverse transcribed with 5 pmol TGIRT-III (InGex) in 100 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 450 mM NaCl, 5 mM MgCl2, 5 mM DTT, 1 mM dNTPs, and 1.25 pmol primer (ocj485) at 60 °C for 1 hr. After the elimination of template RNAs by alkali treatment, cDNA was purified on a 10 % TBE-Urea polyacrylamide gel. The single stranded cDNA was then circularized using 50 U of CircLigase II(Epicenter) at 60 °C for 1 hr, followed by addition of another 50 U of CircLigase II for an additional 1 hr at 60 °C. cDNA was amplified using Phusion DNA polymerase (New England Biolabs) with o231 primer and index primers. After 12-18 rounds of PCR amplification, the product was gel purified from an 8 % TBE-Urea polyacrylamide gel (Thermo Fisher Scientific).
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| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina MiSeq |
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| Description |
total tRNA
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| Data processing |
3’ linker sequences and one nucleotide at the 5’ end was trimmed
Bowtie v. 1.2.2 with default settings was used for mapping reads to reference tRNA sequences retrieved from tRNAdb (http://trnadb.bioinf.uni-leipzig.de/DataOutput/Search)
Two sequences in V. cholerae (tdbD00003706, tdbD00008082) and three sequences in E. coli (tdbD00007320, tdbD00010329, and tdbD00011810) were eliminated from the reference sequences due to extremely low coverage.
Mpileup files were made using the samtools mpileup command without any filtration (option, -A –ff 4 -x -B -q 0 -d 10000000 -f).
The frequency of misincorporation was calculated in each mpileup file. 5’ end termini of the mapped reads were piled up using the bedtools genomecov command (-d -5 -ibam).
To calculate the termination frequency, the number of 5’ termini at any given position was divided by the total number of mapped termini at the given position along with all upstream positions (5’ side).
genome build: Sequencing data of Vibrio cholerae and Escherichia coli are mapped to VCtRNA.fa and ECtRNAs.fa, respectively. These reference files are available at https://drive.google.com/open?id=1EsilghrGGcKp2cwND6Q1oPLk_cI2cWww
processed data files format and content: Tab-delimited text files containing frequencies of RT-signatures (misincorporation and termination) observed in tRNAs. Each row and column represents species and positions of tRNAs, respectively.
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| Submission date |
Mar 27, 2020 |
| Last update date |
May 15, 2020 |
| Contact name |
Satoshi Kimura |
| E-mail(s) |
s.kimura.res@gmail.com
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| Organization name |
Brigham and Women's Hospital
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| Lab |
Waldor Lab
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| Street address |
181 Longwood Avenue
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| City |
Boston |
| State/province |
Massachusetts |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL21022 |
| Series (1) |
| GSE147614 |
Comparative tRNA-seq for rapid profiling of tRNA modifications in a non-model organism |
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| Relations |
| BioSample |
SAMN14466770 |
| SRA |
SRX8010172 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4435459_VC_miaA_24h.txt.gz |
36.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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