|
| Status |
Public on Jan 07, 2021 |
| Title |
ShZc3h10 3 |
| Sample type |
SRA |
| |
|
| Source name |
C3H10T1/2 preadipocytes
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Mesenchymal stem cells
passage: 3
differentiated: Adipocytes
transduced: ShZc3h10
|
| Treatment protocol |
Cells were transduced 24h after congluency and induced to differentiate with DMIR cocktail (Dexamethasone 2µg/ml, 3-isobutyl-1-methylxanthine (IBMX) 0.5mM, insulin 5µg/ml and rosiglitazone 5µM)
|
| Growth protocol |
Cells were grown in DMEM high glucosesuppemented with 10% FBS, 1% Pen/Strep, 1% Gln
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For preparation of 4sU-RNAseq libraries from Scramble and ShZc3h10 36h differentiated pradipocytes. 4-thiouridine (4sU, T4509 Sigma-Aldrich) was added to the medium at a final concentration of 300uM for 10 min. before harvesting. The labeled RNA was isolated and processed as described (Austenaa et al., 2015). The nascent 4sU-labelled RNA was extracted from 50 ug of total TRIZOL-isolated RNA.
100-200 ng of the isolated nascent 4sU-labelled RNA was used for cDNA-library synthesis using the QIAseq Stranded Total RNA Library Kit (Qiagen).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Adapter Trimming using bbduk.sh e.g. "bbduk.sh -Xmx1g threads=7 in=<<Sample.fastq>> out=<<Sample.trim.fastq>> ref=<<adapters.fa>> ktrim=r k=28 mink=13 hdist=1 tbo "
Mapping to reference mm10 using ncbiRefSeqCurated annotation 2017-11-16 e.g. "tophat2 -p 4 -G <<Refseq_Curated>> -o <<Sample_name>> --max-multihits 2 --b2-very-sensitive <<Bowtie2_Index>> <<Sample.fastq>>"
Select uniquely mapped reads e.g. "samtools view -h -F 4 <<accepted_hits.bam>> | grep -v "NH:i:2" | samtools view -b -S -"
Remove ENCODE black list reagions using the Boyle lab list https://github.com/Boyle-Lab/Blacklist e.g. "bedtools intersect -a <<Aligned.bam>> -b <<Encode_black.list.bed>> -v > <<Cleaned_Sample.bam>>"
Creationof strand specific BigWig e.g. "bamCoverage -p 20 -bs 1 --scaleFactor <<Precomputed_scale_factor>> --filterRNAstrand <<forward or Reward>> -b <<Sample.bam>> -o <<Out_folder>>"
Genome_build: mm10
Supplementary_files_format_and_content: .bw are BigWig normalised files
Supplementary_files_format_and_content: .bam are BAM format files
|
| |
|
| Submission date |
Apr 01, 2020 |
| Last update date |
Jan 08, 2021 |
| Contact name |
Nico Mitro |
| E-mail(s) |
[email protected]
|
| Phone |
00390250318344
|
| Organization name |
Università degli studi di Milano
|
| Department |
Pharmacological and biomolecular sciences
|
| Street address |
Via Balzaretti 9
|
| City |
Milan |
| State/province |
MI |
| ZIP/Postal code |
20133 |
| Country |
Italy |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE147941 |
Zc3h10 controls pre-mRNA profile in preadipocytes |
|
| Relations |
| BioSample |
SAMN14521614 |
| SRA |
SRX8044299 |
