|
| Status |
Public on Aug 19, 2019 |
| Title |
R2e - DNase treatment, deacylation, and oxidation |
| Sample type |
SRA |
| |
|
| Source name |
DO treatment, B replicate
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
strain: K-12
substrain: MG1655
|
| Treatment protocol |
Cells were not treated
|
| Growth protocol |
grown at 37°C in standard LB medium, sampling at an OD 600 of ~4.0
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with a standard trizol protocol. Total RNA was either not treated (nD), or treated with DNase (NT), treated with DNase and deacylated (D), treated with DNase, oxydized an deacylated (OD), or treated with DNase, deacylated and oxydized (DO) prior to 3' adapter ligation. After reverse transcription, chimeric RNA/cDNA products were directly loaded on gel for size selection between 35 to 200 bp by electrophoresis, to eliminate the excess of 3’ adapters and select mid-size RNA transcripts.
Gel-extracted products were subjected to poly-A tailing by a TdT enzyme. A first round of selective pre-amplification of the cDNA was achieved with Illumina TruSeq sRNA RTP with TGG overhang and Illumina TruSeq sRNA 5’ adapter primer with polyT(20). A second amplification with Illumina RP1 and Illumina TruSeq sRNA Index primer (RPIX) that added a specific tag to each of the samples provided the final libraries.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
tRNA epitranscriptome treated with DNase, deacylated, and oxidated (DdO condition)
|
| Data processing |
Library strategy: Dts-seq
Remove adapters and trim poly-A tails from R2 reads using cutadapt (version 1.11, with –adapter and --minimum-length options)
Map on E. coli genome using Bowtie2 (version 2.2.8, with –local option)
Filter mapped reads and with a CCA sequence motif at the 3’ end (awk command line ; see https://github.com/i2bc/dts-seq)
Compute stranded genomic coverages using samtools (version 1.4, depth command with -a and -d options)
Compute the termination signal for tRNA using a dedicaced R code (see https://github.com/i2bc/dts-seq)
Genome_build: GCF_000005845.2_ASM584v2
Supplementary_files_format_and_content: The txt files contain the number of reads at each genomic base position, the first column is for the forward strand while the second column is for the reverse strand.
|
| |
|
| Submission date |
Aug 18, 2019 |
| Last update date |
Jun 16, 2020 |
| Contact name |
Claire Toffano-Nioche |
| E-mail(s) |
[email protected]
|
| Organization name |
CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay
|
| Department |
UFR des Sciences
|
| Lab |
Institute for Integrative Biology of the Cell (I2BC)
|
| Street address |
Avenue de la Terrasse, b. 21
|
| City |
Gif-sur-Yvette |
| ZIP/Postal code |
91198 |
| Country |
France |
| |
|
| Platform ID |
GPL18956 |
| Series (1) |
| GSE135937 |
Dts-seq: a simple method of library preparation for a highly reproducible characterization of the tRNA epitranscriptome by deep sequencing |
|
| Relations |
| BioSample |
SAMEA5747344 |
| SRA |
ERX3431639 |
