|
| Status |
Public on Nov 20, 2024 |
| Title |
HEK293T (SLAM-Seq), STM2457, no4SU, rep2 |
| Sample type |
SRA |
| |
|
| Source name |
HEK293T
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
cell type: Human embryonic kidney 293T cell line
genotype: WT
treatment: STM2457
|
| Treatment protocol |
HEK293T cells were seeded in 24 h well plate. At 80% confluency, cells were treated with STM2457 inhibitor or DMSO. After 3h, the metabolic labeling was performed by addition of 100 µM s4U.
|
| Growth protocol |
HEK293T cells were grown and cultured in DMEM supplemented with 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin/streptomycin and were incubated at 37 °C with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After incubation, cells were directly lysed in TRIzol (Thermo Fisher Scientific, #15596026) reagent in reducing conditions. RNA was resuspended in the elution buffer provided in the Lexogen anabolic kit. Exactly 5 µg of RNA was subjected to iodoacetamide treatment.
NGS library prep was performed with Lexogen`s QuantSeq 3´mRNA-Seq V2 Library Prep Kit FWD with Unique Dual Indices (12nt) following Lexogen`s standard protocol (0191UG444V0111). Libraries were prepared with a starting amount of 200ng (except sample 7 with 59ng) and amplified in 15 PCR cycles. Libraries were profiled in a High Sensitivity DNA on a 2100 Bioanalyzer (Agilent technologies) and quantified using the Qubit 1x dsDNA HS Assay Kit, in a Qubit 4.0 Fluorometer (Life technologies). All 24 samples were pooled in equimolar ratio and sequenced on 1 NextSeq2000 P3 (100cycles) FC,SR for 1x 114 cycles plus 2x 12 cycles for the dual index read.
SLAM-seq with Lexogen`s QuantSeq 3´mRNA-Seq V2 Library Prep Kit FWD
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 2000 |
| |
|
| Data processing |
Quality control: FastQC (v0.11.9)
Mapping: STAR (v2.7.8a) --runThreadN 16 --outSAMtype BAM SortedByCoordinate --outFilterMultimapNmax 1 --outSAMattributes All --readFilesCommand zcat --alignEndsType EndToEnd
SLAMseq data process: GRAND-SLAM (v2.0.5f) -e Slam -allGenes -progress -plot -D -minEstimateReads 1000
Using grandR (v0.2.5) to calculate the half lives.
Assembly: GRCh38.p12
Supplementary files format and content: txt half lives data (per gene)
|
| |
|
| Submission date |
Jul 26, 2024 |
| Last update date |
Nov 20, 2024 |
| Contact name |
Julian Konig |
| E-mail(s) |
[email protected]
|
| Organization name |
Institute of Molecular Biology
|
| Street address |
Ackermannweg 9
|
| City |
Mainz |
| ZIP/Postal code |
55128 |
| Country |
Germany |
| |
|
| Platform ID |
GPL30173 |
| Series (2) |
| GSE248575 |
m6A sites in the coding region trigger translation-dependent mRNA decay. |
| GSE273217 |
m6A sites in the coding region trigger translation-dependent mRNA decay (SLAM-seq) |
|
| Relations |
| BioSample |
SAMN42839184 |
| SRA |
SRX25486389 |
