|
| Status |
Public on Sep 21, 2022 |
| Title |
rS6: MEF_Fbxo4(-/-)+non_targeting siRNA rep 2 [RPF] |
| Sample type |
SRA |
| |
|
| Source name |
C57BL/6
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: Murine Embrionic Fibroblasts
genotype: Fbxo4 knock-out
treatment: control RNAi
|
| Treatment protocol |
Fbxo4(+/+) or Fbxo4(-/-) cells were transfected with either non-targeting (Dharmacon-Horizon, cat#D-001810-01-20) or anti-hnRNPK siRNA (Dharmacon-Horizon, cat#L-048002-01-0020). RNAi was delivered by Lipofectamine RNAiMAX (Thermofisher) according to the manufacturer guide.
|
| Growth protocol |
Murine Embryonic Fibroblasts (MEFs) were cultured in DMEM (Corning) media supplemented with 10% FBS (Gemini) and Penicillin-Streptomycin (Corning)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell extract was treated with MNase, CaCl2 (5mM final concentration), and Turbo DNAse I and incubated at 25°C for 30 minutes; the digestion was stopped with SUPERase-inhibitor and placing on ice.
The cell extract was loaded onto a 15%-45% sucrose gradient to separate the polysomes by ultra-centrifugation. The relevant fractions corresponding to the 80S monosome were collected and precipitated using ethanol and the RNA was isolated
Total RNA was isolated from each sample and subjected to the Turbo DNase I digestion and then RNA-seq libraries. The ribosome-protected fragments were subjected to denaturing polyacrylamide-mediated gel electrophoresis and were gel-extracted based on size (26-mers to 34-mers) followed by rRNA depletion by RiboMinus kit (Ambion)
For total RNA sequencing, libraries were constructed using Illumina’s TruSeq Stranded Total RNA kit. The ribosome protected fragment library was generated using NEXT-Flex smRNA-Seq Kit v3 (Perkin Elmer)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
RNA sequencing & RPF sequencing in separate raw files
|
| Data processing |
Basecalling with Ilumina Casava software
FASTQ Quality Filter module of FASTX-Toolkit
Read collapsing by UMI by Collapse module
UMI trimming by Cutadapt
Alignment to mm10 by STAR v2.5.3a
Bam file proccessd by RIboProfiling package v1.2.2
Coverage counts on coding region (CDS) obtained by RxDb.Mmusculus.UCSC.mm10.knownGene v3.10.0
DEseq2 pipeline version 1.26.0 for differential expressed gene analysis
Genome_build: mm10
Supplementary_files_format_and_content: Sample coding for RNA and RPF; cont_tab_sample.txt
Supplementary_files_format_and_content: normalized count from DEseq2; table_norCount_RNAandRPF.txt
Supplementary_files_format_and_content: raw count for total RNA sequencing; summary_gene_count_rpf_table.txt
Supplementary_files_format_and_content: raw count for RPF sequencing; summary_gene_count_rna_table.txt
|
| |
|
| Submission date |
Feb 10, 2022 |
| Last update date |
Sep 21, 2022 |
| Contact name |
J Alan Diehl |
| E-mail(s) |
jad283@case.edu
|
| Phone |
2163680268
|
| Organization name |
Case Western Reserve University
|
| Department |
Department of Biochemistry
|
| Lab |
Deihl Lab
|
| Street address |
2109 Adelbert Rd. SOM Wood/W402
|
| City |
Cleveland |
| State/province |
Ohio |
| ZIP/Postal code |
44106 |
| Country |
USA |
| |
|
| Platform ID |
GPL21626 |
| Series (1) |
| GSE196483 |
Ribosome profiling of Murine Embryonic Fibroblasts in the context of different Fbxo4 and hnRNPK status. |
|
| Relations |
| BioSample |
SAMN25829766 |
| SRA |
SRX14127412 |
