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| Status |
Public on Jul 26, 2019 |
| Title |
TCPS_H2 |
| Sample type |
SRA |
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| Source name |
HUVEC cultivated on TCPS
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| Organism |
Homo sapiens |
| Characteristics |
sample type: HUVEC obtained from the second donor cultivated on TCPS
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| Treatment protocol |
Umbilical vein was washed with 50 ml of phosphate buffer (PBS), then with 20 ml of collagenase IV buffer (1.5 mM HEPES, 14 mM NaCl, 0.4 mM KCl, 0.12 mM CaCl2, 0.04 mM MgSO4, 0.76 mM D-glucose, pH 7.4), and incubated for 15 min at 37°C with 0.1 % collagenase IV (Gibco, USA) in collagenase IV buffer. After incubation, supernatant with detached cells was collected, vein was additionally washed with 20 ml PBS, both solutions were combined and centrifuged at 800g for 10 minutes to pellet cells. Cells were resuspended and cultivated in IMDM (Gibco, USA) with 10% FBS (Gibco, USA) and antibiotics. The next day adherent cells were carefully washed with IMDM to remove blood cells and further cultivated in fresh IMDM with 10% FBS (passage 0). The cells were passaged with 0.1% solution of collagenase IV once per 3-4 days after 70-80% confluence was achieved. Cells from the 2-nd passage were seeded on the surface of dUV and 3D matrices. To obtain synthetic scaffolds, discs (d = 16 mm) were cut out of matrix specimens and dUV fragments, placed in a 24-well plate and pressed down with polytetrafluoroethylene rings (outer and inner diameters, 16 and 12 mm). Scaffolds in plates were treated with 25 kGy electron beam irradiation using ILU-6 accelerator (INP, Russia). Before seeding with cells scaffolds were pre-incubated with IMDM for 2 hours. HUVEC were seeded onto scaffolds in density of 8×103 cells per each plate well. After 12 hours non-adhered cells were removed, fresh culture medium was added and cells were cultivated for 48 hours. Control cells (TCPS) were cultivated in wells without any matrices.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
For gene expression analysis HUVEC cells from 3 different biological donors were used. HUVEC cells were seeded on surface scaffolds as described above, after 48 hours culture medium was removed, matrices were washed with one change of phosphate buffer and transferred to tubes with 750 μl of TriZol reagent (Life Technology, USA). RNA samples were isolated according to the protocol supplied by the manufacturer. RNA was precipitated with 70% alcohol in the presence of 40 µg glycogen (Thermo Scientific, Lithuania): the precipitate was air dried, dissolved in RNA storage medium (Sigma, Germany) and then dried on a vacuum rotary evaporator.
The libraries were prepared for the sequencing using Lexogen QuantSeq 3’ mRNA-Seq Library Prep Kit (Lexogen, Austria) and were sequenced with Illumina HiSeq3000
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
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| Data processing |
Quality control and filtering of raw data was performed using the FastQC and FASTQ Quality Filter (-q 20 –p 75) from FASTX-toolkit (http://hannonlab.cshl.edu/fastx_toolkit/)
According to library preparation kit recommendations, 12 bp segments of raw reads were trimmed out with FASTQ Trimmer from FASTX-toolkit
RNA-seq reads were aligned to NCBI GRCh38 genome (https://support.illumina.com/sequencing/sequencing_software/igenome.html) using HISAT2 aligner (--score-min L,0,-0.5)
Reads corresponding to rRNA and tRNA were removed using split_bam tool from RSeQC package and masked annotation file
Reads aligned to genomic locations were counted using FeatureCounts tool from the Subread package. Differential expression analysis was performed with DESeq2
Genome_build: NCBI GRCh38
Supplementary_files_format_and_content: tabular files were generated with featureCounts and contain raw gene counts
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| Submission date |
Jul 25, 2019 |
| Last update date |
Jul 27, 2019 |
| Contact name |
Petr Laktionov |
| E-mail(s) |
laktionov@mcb.nsc.ru
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| Organization name |
Institute of molecular and cellular biology SD RAS
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| Lab |
Genomics lab
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| Street address |
Lavrenitev ave 8/2
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| City |
Novosibirsk |
| ZIP/Postal code |
630090 |
| Country |
Russia |
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| Platform ID |
GPL21290 |
| Series (1) |
| GSE134909 |
Comparative gene expression profiling of human primary endotheliocytes cultivated on polyurethane-based electrospun 3D matrices and natural decellularized vein |
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| Relations |
| BioSample |
SAMN12367646 |
| SRA |
SRX6595803 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3978706_TCPS_H2_counts.tab.gz |
3.1 Mb |
(ftp)(http) |
TAB |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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