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| Status |
Public on Jan 20, 2017 |
| Title |
Mutant2_2 |
| Sample type |
SRA |
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| Source name |
Mutant2_2
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| Organism |
Homo sapiens |
| Characteristics |
cell type: WA09 Human Embryonic Stem Cell
genotype/variation: a single-copy deletion of chr17p13.1
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| Treatment protocol |
Single-cell cloning was used to derive pure subpopulations of mutant and wild-type cells from the original mosaic MefEnz culture. MefEnz cells cultured on irradiated mouse embryonic fibroblasts (MEFs) in standard hPSC medium, consisting of Dulbecco’s modified eagle medium DMEM/F12 (Life Technologies) with 20% Knockout Serum Replacement (Life Technologies), 1 mM GlutaMAX (Life Technologies), 0.1 mM nonessential amino acids (NEAA, Life Technologies), 0.1 mM -mercaptoethanol, and 12 ng/ml basic FGF (Life Technologies), were treated for 1 hour with 10 M ROCK inhibitor Y-27632 (Calbiochem) before dissociation. The medium was supplemented with SMC4 (0.58 l SMC4/ 1 ml medium) (BD Biosciences) for one day.
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| Growth protocol |
The four WA09 clones (2 wild-type and 2 mutant) were initially grown on MEF feeder layers in standard hPSC medium. These initial cultures were transitioned to feeder-free conditions in a stepwise fashion by passaging them with Accutase onto Geltrex (Gibco) with MEF-conditioned medium (MEF-CM) supplemented with 12 ng/ml basic FGF. After one passage in MEF-CM, the cultures were either continued in MEF-CM or passaged into E8 medium (Life Technologies). For all cultures, cells were grown at 37°C and 5% CO2 and passaged every three to four days, when they were ~80% confluent. At each passage, 150,000 cells were seeded per well of a 6-well plate (equivalent to a cell plating density of ~15,600 cells/ cm2). The medium was changed daily.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using the mirVana miRNA Isolation Kit (Life Technologies, Inc.). The RNA was then quantified (Qubit RNA Assay Kit, Life Technologies, Inc.), and quality controlled (RNA6000 Nano Kit and BioAnalyzer 2100, Agilent).
Approximately 500 ng was used as input for the Illumina TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, Inc.) and sequencing libraries were created according to the manufacturer’s protocol. Briefly, poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads. Following purification, the mRNA was fragmented and copied into first strand cDNA using random primers and reverse transcriptase. Second strand cDNA synthesis was then done using DNA Polymerase I and RNase H. The cDNA was then ligated to adapters and enriched with PCR to create the final cDNA library. The library was then pooled and sequenced on a HiSeq 2500 (Illumina, Inc.) instrument as per manufacturer’s instructions. Sequencing was performed up to 2 X 101 cycles.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Fastq files were quality controlled using FastQC (Babraham Bioinformatics) and any adapter sequences, as well as twelve bases from the five prime end, were removed and mapped to hg19 using STAR (v. 2.4.0a
Samtools (version 0.1.19) was then used to sort, merge, and eliminate duplicate reads
Expression levels for each gene were quantified using the python script rpkmforgenes and annotated using RefSeq (archive-2012-03-09-03-24-410). Genes that did not have at least one sample with at least ten reads were removed from the analysis.
The gene count file was then normalized and differential expression was performed by the R (v.3.0.1) package DESeq (v.1.16.0)
Transcripts with an adjusted P-Value less than 0.05 and an absolute log fold change of more than 1.2 were considered differentially expressed.
All KEGG pathway enrichment analysis was performed using DAVID Bioinformatics Resource 6.7 (http://david.abcc.ncifcrf.gov/home.jsp)
Genome_build: hg19
Supplementary_files_format_and_content: .xlsx including DESeq normalized counts filtered for genes with at least 10 reads in at least one sample
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| Submission date |
Jul 14, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Robert E Morey |
| E-mail(s) |
robmoreyucsd@gmail.com, remorey@health.ucsd.edu
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| Organization name |
UCSD
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| Department |
Pathology
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| Lab |
Parast
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| Street address |
2880 Torrey Pines Scenic Dr
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE70874 |
Spontaneous single-copy loss of TP53 in human embryonic stem cells markedly increases cell proliferation and survival [RNA-Seq] |
| GSE70875 |
Spontaneous single-copy loss of TP53 in human embryonic stem cells markedly increases cell proliferation and survival |
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| Relations |
| BioSample |
SAMN03857593 |
| SRA |
SRX1094559 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data are available on Series record |
| Raw data are available in SRA |
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