18 research outputs found
DataSheet1_Gene-centric coverage of the human liver transcriptome: QPCR, Illumina, and Oxford Nanopore RNA-Seq.PDF
It has been shown that the best coverage of the HepG2 cell line transcriptome encoded by genes of a single chromosome, chromosome 18, is achieved by a combination of two sequencing platforms, Illumina RNA-Seq and Oxford Nanopore Technologies (ONT), using cut-off levels of FPKM > 0 and TPM > 0, respectively. In this study, we investigated the extent to which the combination of these transcriptomic analysis methods makes it possible to achieve a high coverage of the transcriptome encoded by the genes of other human chromosomes. A comparative analysis of transcriptome coverage for various types of biological material was carried out, and the HepG2 cell line transcriptome was compared with the transcriptome of liver tissue cells. In addition, the contribution of variability in the coverage of expressed genes in human transcriptomes to the creation of a draft human transcriptome was evaluated. For human liver tissues, ONT makes an extremely insignificant contribution to the overall coverage of the transcriptome. Thus, to ensure maximum coverage of the liver tissue transcriptome, it is sufficient to apply only one technology: Illumina RNA-Seq (FPKM > 0).</p
DataSheet2_Gene-centric coverage of the human liver transcriptome: QPCR, Illumina, and Oxford Nanopore RNA-Seq.xlsx
It has been shown that the best coverage of the HepG2 cell line transcriptome encoded by genes of a single chromosome, chromosome 18, is achieved by a combination of two sequencing platforms, Illumina RNA-Seq and Oxford Nanopore Technologies (ONT), using cut-off levels of FPKM > 0 and TPM > 0, respectively. In this study, we investigated the extent to which the combination of these transcriptomic analysis methods makes it possible to achieve a high coverage of the transcriptome encoded by the genes of other human chromosomes. A comparative analysis of transcriptome coverage for various types of biological material was carried out, and the HepG2 cell line transcriptome was compared with the transcriptome of liver tissue cells. In addition, the contribution of variability in the coverage of expressed genes in human transcriptomes to the creation of a draft human transcriptome was evaluated. For human liver tissues, ONT makes an extremely insignificant contribution to the overall coverage of the transcriptome. Thus, to ensure maximum coverage of the liver tissue transcriptome, it is sufficient to apply only one technology: Illumina RNA-Seq (FPKM > 0).</p
DataSheet4_Gene-centric coverage of the human liver transcriptome: QPCR, Illumina, and Oxford Nanopore RNA-Seq.xlsx
It has been shown that the best coverage of the HepG2 cell line transcriptome encoded by genes of a single chromosome, chromosome 18, is achieved by a combination of two sequencing platforms, Illumina RNA-Seq and Oxford Nanopore Technologies (ONT), using cut-off levels of FPKM > 0 and TPM > 0, respectively. In this study, we investigated the extent to which the combination of these transcriptomic analysis methods makes it possible to achieve a high coverage of the transcriptome encoded by the genes of other human chromosomes. A comparative analysis of transcriptome coverage for various types of biological material was carried out, and the HepG2 cell line transcriptome was compared with the transcriptome of liver tissue cells. In addition, the contribution of variability in the coverage of expressed genes in human transcriptomes to the creation of a draft human transcriptome was evaluated. For human liver tissues, ONT makes an extremely insignificant contribution to the overall coverage of the transcriptome. Thus, to ensure maximum coverage of the liver tissue transcriptome, it is sufficient to apply only one technology: Illumina RNA-Seq (FPKM > 0).</p
DataSheet3_Gene-centric coverage of the human liver transcriptome: QPCR, Illumina, and Oxford Nanopore RNA-Seq.docx
It has been shown that the best coverage of the HepG2 cell line transcriptome encoded by genes of a single chromosome, chromosome 18, is achieved by a combination of two sequencing platforms, Illumina RNA-Seq and Oxford Nanopore Technologies (ONT), using cut-off levels of FPKM > 0 and TPM > 0, respectively. In this study, we investigated the extent to which the combination of these transcriptomic analysis methods makes it possible to achieve a high coverage of the transcriptome encoded by the genes of other human chromosomes. A comparative analysis of transcriptome coverage for various types of biological material was carried out, and the HepG2 cell line transcriptome was compared with the transcriptome of liver tissue cells. In addition, the contribution of variability in the coverage of expressed genes in human transcriptomes to the creation of a draft human transcriptome was evaluated. For human liver tissues, ONT makes an extremely insignificant contribution to the overall coverage of the transcriptome. Thus, to ensure maximum coverage of the liver tissue transcriptome, it is sufficient to apply only one technology: Illumina RNA-Seq (FPKM > 0).</p
Targeted Quantitative Screening of Chromosome 18 Encoded Proteome in Plasma Samples of Astronaut Candidates
This
work was aimed at estimating the concentrations of proteins
encoded by human chromosome 18 (Chr 18) in plasma samples of 54 healthy
male volunteers (aged 20–47). These young persons have been
certified by the medical evaluation board as healthy subjects ready
for space flight training. Over 260 stable isotope-labeled peptide
standards (SIS) were synthesized to perform the measurements of proteins
encoded by Chr 18. Selected reaction monitoring (SRM) with SIS allowed
an estimate of the levels of 84 of 276 proteins encoded by Chr 18.
These proteins were quantified in whole and depleted plasma samples.
Concentration of the proteins detected varied from 10<sup>–6</sup> M (transthyretin, P02766) to 10<sup>–11</sup> M (P4-ATPase,
O43861). A minor part of the proteins (mostly representing intracellular
proteins) was characterized by extremely high inter individual variations.
The results provide a background for studies of a potential biomarker
in plasma among proteins encoded by Chr 18. The SRM raw data are available
in ProteomeXchange repository (PXD004374)
State of the Art of Chromosome 18-Centric HPP in 2016: Transcriptome and Proteome Profiling of Liver Tissue and HepG2 Cells
A gene-centric approach
was applied for a large-scale study of
expression products of a single chromosome. Transcriptome profiling
of liver tissue and HepG2 cell line was independently performed using
two RNA-Seq platforms (SOLiD and Illumina) and also by Droplet Digital
PCR (ddPCR) and quantitative RT-PCR. Proteome profiling was performed
using shotgun LC–MS/MS as well as selected reaction monitoring
with stable isotope-labeled standards (SRM/SIS) for liver tissue and
HepG2 cells. On the basis of SRM/SIS measurements, protein copy numbers
were estimated for the Chromosome 18 (Chr 18) encoded proteins in
the selected types of biological material. These values were compared
with expression levels of corresponding mRNA. As a result, we obtained
information about 158 and 142 transcripts for HepG2 cell line and
liver tissue, respectively. SRM/SIS measurements and shotgun LC–MS/MS
allowed us to detect 91 Chr 18-encoded proteins in total, while an
intersection between the HepG2 cell line and liver tissue proteomes
was ∼66%. In total, there were 16 proteins specifically observed
in HepG2 cell line, while 15 proteins were found solely in the liver
tissue. Comparison between proteome and transcriptome revealed a poor
correlation (<i>R</i><sup>2</sup> ≈ 0.1) between
corresponding mRNA and protein expression levels. The SRM and shotgun
data sets (obtained during 2015–2016) are available in PASSEL
(PASS00697) and ProteomeExchange/PRIDE (PXD004407). All measurements
were also uploaded into the in-house Chr 18 Knowledgebase at http://kb18.ru/protein/matrix/416126
Chromosome 18 Transcriptome Profiling and Targeted Proteome Mapping in Depleted Plasma, Liver Tissue and HepG2 Cells
The final goal of the Russian part of the Chromosome-centric
Human
Proteome Project (C-HPP) was established as the analysis of the chromosome
18 (Chr 18) protein complement in plasma, liver tissue and HepG2 cells
with the sensitivity of 10<sup>–18</sup> M. Using SRM, we have
recently targeted 277 Chr 18 proteins in plasma, liver, and HepG2
cells. On the basis of the results of the survey, the SRM assays were
drafted for 250 proteins: 41 proteins were found only in the liver
tissue, 82 proteins were specifically detected in depleted plasma,
and 127 proteins were mapped in both samples. The targeted analysis
of HepG2 cells was carried out for 49 proteins; 41 of them were successfully
registered using ordinary SRM and 5 additional proteins were registered
using a combination of irreversible binding of proteins on CN-Br Sepharose
4B with SRM. Transcriptome profiling of HepG2 cells performed by RNAseq
and RT-PCR has shown a significant correlation (<i>r</i> = 0.78) for 42 gene transcripts. A pilot affinity-based interactome
analysis was performed for cytochrome b5 using analytical and preparative
optical biosensor fishing followed by MS analysis of the fished proteins.
All of the data on the proteome complement of the Chr 18 have been
integrated into our gene-centric knowledgebase (www.kb18.ru)
Chromosome 18 Transcriptome Profiling and Targeted Proteome Mapping in Depleted Plasma, Liver Tissue and HepG2 Cells
The final goal of the Russian part of the Chromosome-centric
Human
Proteome Project (C-HPP) was established as the analysis of the chromosome
18 (Chr 18) protein complement in plasma, liver tissue and HepG2 cells
with the sensitivity of 10<sup>–18</sup> M. Using SRM, we have
recently targeted 277 Chr 18 proteins in plasma, liver, and HepG2
cells. On the basis of the results of the survey, the SRM assays were
drafted for 250 proteins: 41 proteins were found only in the liver
tissue, 82 proteins were specifically detected in depleted plasma,
and 127 proteins were mapped in both samples. The targeted analysis
of HepG2 cells was carried out for 49 proteins; 41 of them were successfully
registered using ordinary SRM and 5 additional proteins were registered
using a combination of irreversible binding of proteins on CN-Br Sepharose
4B with SRM. Transcriptome profiling of HepG2 cells performed by RNAseq
and RT-PCR has shown a significant correlation (<i>r</i> = 0.78) for 42 gene transcripts. A pilot affinity-based interactome
analysis was performed for cytochrome b5 using analytical and preparative
optical biosensor fishing followed by MS analysis of the fished proteins.
All of the data on the proteome complement of the Chr 18 have been
integrated into our gene-centric knowledgebase (www.kb18.ru)
Chromosome 18 Transcriptome Profiling and Targeted Proteome Mapping in Depleted Plasma, Liver Tissue and HepG2 Cells
The final goal of the Russian part of the Chromosome-centric
Human
Proteome Project (C-HPP) was established as the analysis of the chromosome
18 (Chr 18) protein complement in plasma, liver tissue and HepG2 cells
with the sensitivity of 10<sup>–18</sup> M. Using SRM, we have
recently targeted 277 Chr 18 proteins in plasma, liver, and HepG2
cells. On the basis of the results of the survey, the SRM assays were
drafted for 250 proteins: 41 proteins were found only in the liver
tissue, 82 proteins were specifically detected in depleted plasma,
and 127 proteins were mapped in both samples. The targeted analysis
of HepG2 cells was carried out for 49 proteins; 41 of them were successfully
registered using ordinary SRM and 5 additional proteins were registered
using a combination of irreversible binding of proteins on CN-Br Sepharose
4B with SRM. Transcriptome profiling of HepG2 cells performed by RNAseq
and RT-PCR has shown a significant correlation (<i>r</i> = 0.78) for 42 gene transcripts. A pilot affinity-based interactome
analysis was performed for cytochrome b5 using analytical and preparative
optical biosensor fishing followed by MS analysis of the fished proteins.
All of the data on the proteome complement of the Chr 18 have been
integrated into our gene-centric knowledgebase (www.kb18.ru)
Chromosome 18 Transcriptome Profiling and Targeted Proteome Mapping in Depleted Plasma, Liver Tissue and HepG2 Cells
The final goal of the Russian part of the Chromosome-centric
Human
Proteome Project (C-HPP) was established as the analysis of the chromosome
18 (Chr 18) protein complement in plasma, liver tissue and HepG2 cells
with the sensitivity of 10<sup>–18</sup> M. Using SRM, we have
recently targeted 277 Chr 18 proteins in plasma, liver, and HepG2
cells. On the basis of the results of the survey, the SRM assays were
drafted for 250 proteins: 41 proteins were found only in the liver
tissue, 82 proteins were specifically detected in depleted plasma,
and 127 proteins were mapped in both samples. The targeted analysis
of HepG2 cells was carried out for 49 proteins; 41 of them were successfully
registered using ordinary SRM and 5 additional proteins were registered
using a combination of irreversible binding of proteins on CN-Br Sepharose
4B with SRM. Transcriptome profiling of HepG2 cells performed by RNAseq
and RT-PCR has shown a significant correlation (<i>r</i> = 0.78) for 42 gene transcripts. A pilot affinity-based interactome
analysis was performed for cytochrome b5 using analytical and preparative
optical biosensor fishing followed by MS analysis of the fished proteins.
All of the data on the proteome complement of the Chr 18 have been
integrated into our gene-centric knowledgebase (www.kb18.ru)