|
| Status |
Public on Mar 01, 2023 |
| Title |
AP-05 |
| Sample type |
SRA |
| |
|
| Source name |
RAW 264.7 cells
|
| Organism |
Mus musculus |
| Characteristics |
replica: Replica1
treatment: anti-HuR antibody+LPS+DMSO treatment
|
| Treatment protocol |
Cells have been harvested after treatments for 6 hours with LPS (1 ug/ml) TMs (10 uM) or DMSO and lysed with 20 mM Tris–HCl at pH 7.5, 100 mM KCl, 5 mM MgCl2, and 0.5% NP-40 supplemented with 1% protease inhibitor cocktail (Sigma Aldrich) and 100 units of RiboLOCK RNase inhibitor (Thermo Fisher Scientific) for 10 min on ice and centrifuged at 15 000 × g for 10 min at 4°C.
|
| Growth protocol |
Murine macrophages RAW264.7 (ICLC, Genova) cell lines were maintained in high glucose Dulbecco’s modified Eagle’s medium (DMEM) by adding 10% fetal bovine serum (FBS, Lonza), 2 mM L-glutamine, 100 U/ml penicillin-streptomycin (Lonza) in standard growth conditions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For RIP-seq,TRIzol reagent was added directly to the beads for HuR-bound RNA isolation and processed for library preparation. For RNA-seq TRIzol reagent was added directly to the cells pellets
RNAs from INPUT and RIP were tested for quality using Bioanalyzer and 7ng of the extracted RNA, having RIN higher than 9, were used for fragmentation at 94 °C for 4 min. RNA libraries were generated using the SMART-Seq Stranded Kit (Takara).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
30 million of RAW 264.7 underwent anti-HuR antibody+LPS+DMSO treatment
AP-05
|
| Data processing |
Raw sequences files’ quality was checked via FastQC (v 0.11.9, http://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
We have performed reads alignment to the mouse reference genome using STAR aligner (Spliced Transcripts Alignment to a Reference, v 2.7.7a)
Counts derive from HT-seq counts integrated in star aligner
Differentially expressed genes (DEGs) were generated using the package DESeq2.
Assembly: Mus musculus GRCm39.103 (GTF file), Mus musculus GRCm39 DNA primary assembly fasta
Supplementary files format and content: Counts from HT-seq counts integrated in star aligner
|
| |
|
| Submission date |
Mar 14, 2022 |
| Last update date |
Mar 01, 2023 |
| Contact name |
Silvano Piazza |
| E-mail(s) |
silvano.piazza@icgeb.org
|
| Organization name |
ICGEB
|
| Lab |
Computational Biology
|
| Street address |
localita' Padriciano 99
|
| City |
Trieste |
| ZIP/Postal code |
34149 |
| Country |
Italy |
| |
|
| Platform ID |
GPL24247 |
| Series (1) |
| GSE198605 |
HuR modulation impairs LPS-response in murine macrophages |
|
| Relations |
| BioSample |
SAMN26658990 |
| SRA |
SRX14491277 |
