|
| Status |
Public on Sep 09, 2020 |
| Title |
MLL4 Q4092X/wt (C6) 24h ATRi [C6_24H_S12] |
| Sample type |
SRA |
| |
|
| Source name |
MLL4 Q4092X/wt (C6) 24h ATRi
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: hTERT-immortalized human mesenchymal stem cells (iMSCs)
genotype/variation: MLL4 Q4092X/+
treatment: 24 hour ATRi treatment
|
| Treatment protocol |
Prior to harvest, cells were treated with 0.2 μM ATR inhibitor for 8 and 2 hours or left untreated, respectively.
|
| Growth protocol |
hTERT-immortalized human mesenchymal stem cells (iMSCs) were cultured at 37°C and 5% CO2 in 1:1 DMEM/F-12 medium (Gibco #11320-074) supplemented with 10% Fetal Bovine Serum (Euroclone #ECS0180L). iMSCs expressing Cas9WT were generated by transducing iMSCs with lentiviral vector generated using the plasmid pCW-Cas9WT (Addgene # 50661). Transduced cells were selected with 1μg/ml of puromycin. The expression of sgRNAs were obtained by transducing iMSCs-Cas9WT with lentiviral vectors generated using the plasmid pLX-sgRNA (Addgene #50662). Transduced cells were selected with 2μg/ml of blasticidin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Wild type and MLL4 Q4092X MSCs were extracted collected by centrifuging cell cultures, either untreated or at 8 and 24 hours after ATR inhibition. Cellular pellets were resuspended with TRIzol (Thermo Fisher cat. #15596026Ambion #15596018), and total RNA was extracted according to the manufacturer’s instructions. Contaminating genomic DNA was removed by DNase (Qiagen cat. #79254) digestion. RNA quality and concentration were assed with the 2100 Bioanalyzer (Agilent cat. #G2939BA) and the Qubit fluorometer (Thermo Fisher cat. # Q33226), respectively.
3’-RNA-seq libraries were prepared by using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen cat. #015.24) and starting from 500 ng of total RNA. Libraries were sequenced as single reads of 50 bp with the Illumina HiSeq2500.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
C6_24H_S12
|
| Data processing |
Sequenced reads quality was assessed using fastQC
Reads were trimmed using Trimmomatic v0.39.
Reads were aligned to the human genome (GRCh38/hg38) using STAR v2.71a.
reads per Kilobase per Megabase of library size (RPKM) were calculated using HOMER v4.10 with the command analyzeRepeats.pl rna hg38 -count 3utr -rpkm -condenseGenes
Rpkm normalized bigwig files created using DeepTools3.1.2.
Genome_build: GRCh38/hg38
Supplementary_files_format_and_content: rpkm-normalized reads per sample are presented in the bigwig-format. RPKM for each gene in all samples are presented in a .txt file.
|
| |
|
| Submission date |
Aug 08, 2019 |
| Last update date |
Sep 09, 2020 |
| Contact name |
CIBIO Zippo |
| E-mail(s) |
alessio.zippo@unitn.it
|
| Organization name |
University of Trento
|
| Department |
Cellular, Computation and Integrative Biology (CIBIO)
|
| Lab |
Chromatin Biology & Epigenetics
|
| Street address |
via sommarive 9
|
| City |
Trento |
| State/province |
Not Applicable |
| ZIP/Postal code |
38123 |
| Country |
Italy |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE135550 |
MLL4 establishes enhancer-associated condensates to counterbalance Polycomb-mediated nuclear mechanical stress in Kabuki Syndrome [3'UTR RNA-seq] |
|
| Relations |
| BioSample |
SAMN12530756 |
| SRA |
SRX6674433 |
