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| Status |
Public on Feb 02, 2024 |
| Title |
r2-MD6-noEU |
| Sample type |
SRA |
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| Source name |
MD6
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MD6
treatment: unlabeled control
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| Treatment protocol |
Cells were treated with 1µM atRA treatment (30min for the pulse samples), for 2h with 1µM atRA for the primed samples and for 2h with 1µM atRA (primed), followed by a short atRA treatment for 30min 36 hours after the priming to obtain the primed + pulse samples.
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| Extracted molecule |
total RNA |
| Extraction protocol |
For the analysis of nascent RNA expression, cells were treated with RA as described above and pulse-labeled with 0.5 mM 5-ethynyluridine for 30 min at 37°C and 5% CO2. After pulse-labeling, total RNA was isolated using TRIzol and purified RNA was further processed using the Click-iTTM Nascent RNA Capture Kit (Invitrogen, C10365)
In brief, 5-EU incorporated RNA was biotinylated, purified and pulled down using Dynabeads™ MyOne™ Streptavidin T1 beads. Before the pulldown, biotinylated custom-made spike-in RNAs #1 (2.5e-5 ug) and #2 (2.5-e-4 ug) (expression vectors kindly provided by Kenneth Zaret, University of Pennsylvania) were added to 1 ug of biotinylated RNA. After washing the beads, cDNA was directly generated off the beads using the Universal Plus Total RNA-Seq with NuQuant kit (Tecan) as described by the manufacturer with the following modifications: fragmentation time was increased to 10 min and after second strand synthesis, beads were collected on a magnet and supernatant was transferred to a new tube for subsequent steps. Quality of the libraries was assessed using the 2100 Bioanalyzer (Agilent cat. #G2939BA) and the Qubit fluorometer (Thermo Fisher cat. #Q33226), pooled and sequenced on an Illumina NovaSeq6000 with 150 bp PE.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
MD6_noEU_2
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| Data processing |
.Raw reads were quality controlled using FastQC and trimmed using Trimmomatic v0.31
Reads were aligned to the human genome hg19 using BowTie2.
SAM files were further processed and duplicates were removed using samtools v1.10
‘MultiBamSummary’ of Deeptools v3.1.2 was used to retrieve the counts per sample over the RefSeq-transcript model in bed format.
Counts transformed to FPKM values and spike-in normalized as described in Palozola et al., 2021)
Assembly: hg19
Supplementary files format and content: count table of spike-in normalized expression values [fpkm]
Supplementary files format and content: bigwig files of merged replicates per treatment
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| Submission date |
Aug 19, 2022 |
| Last update date |
Feb 02, 2024 |
| Contact name |
CIBIO Zippo |
| E-mail(s) |
alessio.zippo@unitn.it
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| Organization name |
University of Trento
|
| Department |
Cellular, Computation and Integrative Biology (CIBIO)
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| Lab |
Chromatin Biology & Epigenetics
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| Street address |
via sommarive 9
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| City |
Trento |
| State/province |
Not Applicable |
| ZIP/Postal code |
38123 |
| Country |
Italy |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE211609 |
Oncogenic enhancers prime quiescent metastatic cells to escape NK immune surveillance by eliciting a transcriptional memory [nascent RNA-seq] |
| GSE211610 |
Oncogenic enhancers prime quiescent metastatic cells to escape NK immune surveillance by eliciting a transcriptional memory |
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| Relations |
| BioSample |
SAMN30409861 |
| SRA |
SRX17148284 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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