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Status |
Public on Sep 09, 2020 |
Title |
MLL4 Q4092X/wt (C6) 8h ATRi [C6_8H_S10] |
Sample type |
SRA |
|
|
Source name |
MLL4 Q4092X/wt (C6) 8h ATRi
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Organism |
Homo sapiens |
Characteristics |
cell type: hTERT-immortalized human mesenchymal stem cells (iMSCs) genotype/variation: MLL4 Q4092X/+ treatment: 8 hour ATRi treatment
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Treatment protocol |
Prior to harvest, cells were treated with 0.2 μM ATR inhibitor for 8 and 2 hours or left untreated, respectively.
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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.
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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.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2500 |
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Description |
C6_8H_S10
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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.
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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] |
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Relations |
BioSample |
SAMN12530758 |
SRA |
SRX6674431 |