|
| Status |
Public on Apr 10, 2024 |
| Title |
wt-replicate D [D] |
| Sample type |
SRA |
| |
|
| Source name |
DU145
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: None
cell line: DU145
cell type: prostate cancer cells
molecule type: small RNA
|
| Treatment protocol |
tRNAs were de-aminoacylated with a solution containing 1mM EDTA and 0.1M Tris-HCl pH 9.0 for 30 min at 37°C. For identifying the m7G deposition position, 1-2 mg of tRNAs were treated with Buffer A (0.2 M Tris-HCl pH, 0.01 M MgCl2, 0.2 M KCl) supplemented with 10 μg of m7GTP (NEB) per 10 μg of tRNAs for 5 min at 85ºC. Freshly prepared NaBH4 was added to a final concentration of 0.5 M and samples were incubated for 30 min on ice. Treated tRNAs were desalted with Micro Bio-Spin 6 columns (Biorad) and ethanol precipitated. To induce complete ꞵ-elimination and cleavage of RNA at methylated guanines, reduced RNAs were treated with aniline/acetate/water solution (1:3:7) (Sigma). All small RNAs were treated with T4 PNK (NEB) to ensure phosphorylated 5’ ends and 3’OH ends, followed by heat inactivation and ethanol precipitation.
|
| Growth protocol |
DU145 were cultured in DMEM with L-Glutamine and pyruvate (Gibco) supplemented with 10% FBS (Gibco) and 1% Penicillin/streptomycin, a humidified atmosphere at 37°C and 5% CO2. Cell cultures were tested for mycoplasma monthly and maintained mycoplasma-free.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cells. Small RNA was size-selected and treated or not treated with NaBH4+Aniline (that induces RNA chain cleavage at methylated guanosines).
Sequencing libraries were prepared following the protocol included with the kit “NEXTflex™ Small RNA-Seq Kit v3,” (©Bioo Scientific Corp. Catalog #5132-05).
|
| |
|
| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Total RNA was extracted from WT (METTL1 expressing) cells. RNAs were not further treated with NaBH4+Aniline.
|
| Data processing |
FASTQs were trimmed for the adapters following the recommendations of the NEXTflex™ Small RNA-Seq Kit manufacturers.
We used bowtie to align the reads against the human genome (GRCh38/UCSC hg38), with the option "-v 2 - m 500" to allow for 2 mismatches and multiple mapping of tRNA fragments, and htseq-count with the "-samout" option to flag alignment reads. tRNA fragments were extracted from the alignment files using custom PERL scripts. Statistical significance of differential expression was evaluated using the R DESeq2 package.
Assembly: GRCh38/UCSC hg38
Supplementary files format and content: read counts and especific tRNAs' sequences
|
| |
|
| Submission date |
May 18, 2022 |
| Last update date |
Apr 10, 2024 |
| Contact name |
Ana Maria Aransay |
| E-mail(s) |
amaransay@cicbiogune.es
|
| Phone |
0034944061325
|
| Organization name |
CIC bioGUNE
|
| Department |
Genome Analysis Platform
|
| Street address |
Parque tecnologico de Bizkaia, Building 801-A
|
| City |
Derio |
| State/province |
BIZKAIA |
| ZIP/Postal code |
48160 |
| Country |
Spain |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE203255 |
Detection of internal m7G modification in the human transcriptome in a quantitative manner and at single-nucleotide resolution |
|
| Relations |
| BioSample |
SAMN28522154 |
| SRA |
SRX15324911 |
