Ploidy assignment and detection of structural variation in K562 cell line
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB74235 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR12799800_1.fastq.gz | Fastqsanger.gz | |||
| ERR12799800_2.fastq.gz | Fastqsanger.gz | |||
| ERR12799900_1.fastq.gz | Fastqsanger.gz | |||
| ERR12799900_2.fastq.gz | Fastqsanger.gz | |||
| ERR12799801_1.fastq.gz | Fastqsanger.gz |
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Project description:RNA-Seq reads and TopHat (Trapnell et al. Bioinformatics 2009) alignments of K562 cell-line transcriptome. These were used to validate the expression of short peptides idenitified by Mass-Spectrometry in K562 cells. K562 polyA+ RNA (Batch 1) and total RNA (batch 2) was purchased from Ambion. We used oligo (dT)-selected polyA+ RNA to construct libraries for RNA-Seq.We then profiled the transcriptome of polyadenylated mRNA-Seq using Illumina sequencing platforms. We then used the sequenced reads to reconstruct the transcriptome using the Cufflinks de-novo assembler (Trapnell et al. Nat.Bio.Tech. 2010). Recent computational and ribosome profiling analyses suggest that many short open reading frames (sORFs) in eukaryotic genomes are translated. However, evidence that these sORFs produce stable polypeptides is lacking. Here we develop a strategy to discover and validate novel sORF-encoded polypeptides (SEPs) in human cells. In total, we detect 117 SEPs, 114 of which are novel, varying in length from 15 to 149 amino acids. Of these, 10 SEPs (0.5%) are derived from long intergenic non-coding RNAs (lincRNAs). We also observe the presence of polycistronic genes and the widespread use of non-AUG start codons, which is a phenomenon historically thought to be rare in the mammalian genome. Quantitative measurements reveal that SEPs can be found at concentrations between ~10-2000 copies per cell, which is within the range of typical cellular proteins. We confirm the translation of a number of these SEPs through heterologous expression of their encoding cDNAs. We also discover that several SEPs possess properties characteristic of functional proteins. These results demonstrate that human sORFs produce numerous stable polypeptides, revealing that the human proteome is larger and more diverse than previously appreciated.
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Project description:This series consists of samples taken from two groups of K562 cells(miR-181a transfected group and control group ) and harvested 48 hours later. We used Agilent human 1A oligo microarray identified the changes in gene expression profile of K562 cells after miR-181a transfection experiment. Further studies aimed to find target mRNA of miR-181a in mammalian cells and its biological function. Experiment Overall Design: The K562 cells in our research were divided into tranfected group and mock-transfected control according to Lim et al. Experiment Overall Design: To transfect, we diluted 60 pmol miR-181 a duplex into 12 µL Opti-Mem each well, then we diluted 3 µL Oligofectamine(Invitrogen) into 48 µL Opti-Mem for each sample, incubated for 5 min at room temperature and add 10 µL of diluted transfection mixture to wells and incubate for another 20 min at room temperature. We next added 100 µL of diluted cells suspension mixture containing 50000 cells on top of the complex. Four hours after transfection, FBS was added to the cells at a final concentration of 10% and the cells were incubated for 48 hours. And control group was transfected only with Oligofectamine but no RNA. Each individual transfection experiment was performed in 10 replicate wells. Experiment Overall Design: After transfection experiments, total RNAs from above two group K562 cells were purified using the RNeasy mini kit (Qiagen, Heidelberg, Germany) in accordance with the manufacturerâs protocols. RNA concentration and purity were estimated spectrophotometrically and the quality further assessed with the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Experiment Overall Design: Total RNA from control group and transfected group were respectively reverse transcription, in vitro transcribed and labeled (Cy3 and Cy5) according to the Agilent Low RNA Input Fluorescent Linear Amplification Kit Protocol, green is control group, red is transfected group. The array hybridization, washing, and scanning procedures were performed in our lab according to Agilent 60-mer oligo microarray processing protocol using 0.75 µg samples of labeled, fragmented cRNA on Agilent Human 1A oligo microarray with 22575 probes (Agilent Technologies). Labeled cRNA is incubated with the microarray in a hybridization chamber at 60ºC for 17 hours, 4 rpm/min. The microarray was scanned using Agilent 2565BA DNA microarray scanner (Agilent Technologies). Data collection was performed by Agilent DNA microarray scanner. Using the Agilent Feature Extraction Software(v.7.5), spots were identified, measured for fluorescence intensity, local background, and arraywide parameters used to normalize the signals. Outliers and saturated spots were rejected according to 1.42xIQR, and local backgroud subtracted from the signals. Signals were then normalized to remove dye bias and arraywide drift in flourescence. Experiment Overall Design: The following two tests are performed to determined the significance of a feature: (1) Log ratio P-value of the red over green processed signals was calculated using two-sided t-test with P<0.01 considered significant, and (2) a boolean flag of 1 indicated that the feature BGSubSignal is well above background and passes the IsPosAndSignif test. Experiment Overall Design: We used Agilent human 1A oligo microarray identified the changes in gene expression profile of K562 cells after miR-181a transfection experiment. Experiment Overall Design: Further studies aimed to find target mRNA of miR-181a in mammalian cells and the function of miR-181a in cancers.
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