|
| Status |
Public on Feb 01, 2023 |
| Title |
DCH1_untreated_biol rep 1 [Ribo-seq] |
| Sample type |
SRA |
| |
|
| Source name |
MB135iDUX4
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MB135iDUX4
cell type: myoblasts
genotype: WT
treatment: untreated
|
| Treatment protocol |
MB135iDUX4 myoblasts were pulsed with or without 1ug/mL doxycycline for 4 hours, incubated for 48 hours, supplemented with or without 50ng/mL IFNg for 16 hours, and harvested at a terminal time point of 68 hours.
Ribo-seq was performed as described previously (Calviello et al., 2016).
|
| Growth protocol |
MB135iDUX4 myoblasts were grown in Ham’s F-10 (Gibco) supplemented with 10% FBS (HyClone), 1% penicillin/streptomycin (Thermo Fisher Scientific), 10ng/mL rhFGF (Promega), 1uM dexamethasone (Sigma), and 3ug/mL puromycin (Sigma)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was harvested using the Direct-zol RNA Miniprep Kit (Zymo Research) following manufacturer's protocols.
RNA-seq libraries were prepared using the NEXTflex Small RNA-Seq Kit v3 (PerkinElmer) following manufacturer's protocols.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
library strategy: Ribo-seq
Clip the 3’ adapter sequence and trim the first and last four bases from the adapter-clipped reads (cutadapt).
Remove rRNA and other small RNA such as tRNA and snoRNA. In this step, we customized the rRNA reference genome from the RNA central database (https://rnacentral.org/) and then used Bowtie2 to align the trimmed reads against it; the unmapped reads are the desirable RPFs. Due to the caveats of maintaining congruent paired-reads after removing rRNA containmanation from forword and reverse reads by Bowtie2, we decided to just use forward reads (R1) for following RPF alignment.
Align the RPFs (R1) to the GRCh38 (p.13) genome built by STAR.
Assess the quality of the mapped RPFs using Bioconductor’s ribosomeProfilingQC package. Here, we calculated the size distribution of the RPFs. It confirmed that most RPFs are congruent with the actual size of ribosomes (26 - 29 nt). We plotted the meta-gene coverage of p-sites densities. It revealed the enrichment of the ribosome footprints at the start codon and, most importantly, shows the trinucleotide footprint periodicity confirming that most p-sites are in-frame with the start codon.
Profile ribosome footprints. Using ribosomeProfilingQC and the Gencode (v35) annotation, we computed the p-sites counts of RPFs of the dominant length (26 - 29 nt) for five different genomic features including the 5’ UTRs, translation start sites (TSS; 13 nucleotides extended up/downstream from the start codon), CDS and 3’ UTRs
Assembly: GRCh38.p13
Supplementary files format and content: Bioconductor's RangesSummarizedExperiment instance (*.rda) of gene counts in CDS region (gencode v35) and a table of non-normalized gene counts (*.csv)
|
| |
|
| Submission date |
Jun 19, 2022 |
| Last update date |
Feb 01, 2023 |
| Contact name |
Stephen Tapscott |
| E-mail(s) |
stapscot@fredhutch.org
|
| Organization name |
Fred Hutch Cancer Research Center
|
| Department |
Human Biology
|
| Lab |
Tapscott
|
| Street address |
1100 Fairview N. Ave
|
| City |
Seattle |
| State/province |
WASHINGTON |
| ZIP/Postal code |
98103 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE206437 |
DUX4 orchestrates translational reprograming by broadly suppressing translation efficiency [Ribo-seq] |
| GSE206439 |
The transcription factor DUX4 orchestrates translational reprogramming by broadly suppressing translation efficiency and promoting expression of DUX4-induced mRNAs |
|
| Relations |
| BioSample |
SAMN29203292 |
| SRA |
SRX15795486 |
