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| Status |
Public on Nov 19, 2018 |
| Title |
3D-CCM3-2hrs |
| Sample type |
SRA |
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| Source name |
HUVEC_3D-CCM3
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HUVEC
sirna for gene knockdown: pdcd10 (LQ-004436-00)
substrate for cultivation: Matrigel
time of incubation: 2 hours
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| Treatment protocol |
Cells were transfected with 50 nM siRNA against human krit1 (LQ-003825-00), ccm2 (LG-014728-01), and pdcd10 (LQ-004436-00), or control non-targeting siRNA (D-001810-01-05), using DharmaFECT 4 transfection reagent. Gene expression was analyzed at 48 hrs, protein expression at 72 hrs after transfection, and phenotypic studies were conducted 60-72 hrs after transfection.
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| Growth protocol |
Human umbilical cord endothelial cells (HUVEC) were maintained in EGM-2 medium at 37°C/5% CO2 and passaged every 3 to 4 days for up to 6 passages at a 1:5 sub-culturing ratio. For tube formation experiments, 4.5-5x103 cells were plated into each well of angiogenesis µ-slides coated with 10 µl of growth factor reduced phenol red-free Matrigel, and incubated for up to 18 hrs.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using RNeasy extraction kit (Qiagen, Valencia, CA, USA) following the manufacturer’s protocol. RNA was reverse-transcribed using the high-capacity cDNA kit (Life Technologies, Carlsbad, CA, USA). The analysis was done StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA), and the level of transcripts was quantified using TaqMan probes (Applied Biosystems): KRIT1 Hs01090981_m1; CCM2 Hs01123856_m1; PDCD10 Hs00200578_m1; TBP Hs00427620_m1. Fold change in gene expression was determined using ΔΔCt algorithm.
HUVEC cells were transfected with non-targeting siRNA or siRNA against krit1, ccm2, and pdcd10 genes as described above. 48 hrs after transfection cells were plated on growth factor reduced Matrigel, and incubated for 2 hrs (initial stages of endothelial tubule formation) or 6 hrs (advanced stages of tubule formation). Total RNA was isolated from each culture (control, and siRNA against krit1, ccm2, and pdcd10) cells cultured on the 2D (Matrigel-coated plastic), and in 3D (Matrigel) substrate, using RNAeasy kit (Qiagen, Valencia, CA, USA) per the manufacturer’s instructions. RNA quality control was performed by studying the relative intensity of 18s and 28s rRNA bands using an Agilent 2100 Bioanalyzer and RNA6000 Pico LabChip Kit (Agilent Technologies). Between 200ng and 1ug of RNA were used directly in the TruSeq mRNA stranded kit (Illumina, San Diego, CA) for isolation of mRNA from total RNA, generation of ds cDNA and preparation of libraries. The cDNA was quantified using the Qubit HS DNA kit (Thermo Fisher Scientific, Rockford, IL), and cDNA libraries were prepared using a TruSeq Stranded mRNA library prep kit (Illumina, San Diego, CA). The samples were run on a NextSeq 500 instrument (Illumina, San Diego, CA) with a read length of PE75bp.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Description |
S11-3D-CCM3-2hrs_S10
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| Data processing |
Quality control and adapter trimming: BBDuk, BBTools package Version 35.92. Values of parameters: ktrim=r k=27 hdist=1 edist=0 mink=4 qtrim=rl trimq=30 minlength=50 qin=33
Transcriptome alignment: TopHat2 v2.1.1 (human genome build GRCh38). Values of parameters: --no-coverage-search --b2-very-sensitive --no-novel-juncs
Reads counting and ranking of differentially expressed genes: GFOLD. Cutoff for generalized fold change: 0.3. Comand for reads counting: gfold count -ann [genome annotation] -tag [SAM file with mapped reads] -o [output file]. Command for identification of differentially expressed genes: gfold diff -s1 [reads counts for sample 1] -s2 [reads counts for sample 2] -suf .read_cnt -o [output_file.diff]
Genome_build: Human genome GRCh38
Supplementary_files_format_and_content: Tab-delimited text files generated by GFOLD, include GeneSymbols, GeneNames, GFOLD values, RPKM values and other information
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| Submission date |
Jun 27, 2018 |
| Last update date |
Nov 20, 2018 |
| Contact name |
Denis Tsygankov |
| E-mail(s) |
denis.tsygankov@bme.gatech.edu
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| Organization name |
Georgia Institute of Technology
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| Department |
Biomedical Engineering
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| Lab |
Integrative Systems Biology
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| Street address |
North Ave NW
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| City |
Atlanta |
| State/province |
GA |
| ZIP/Postal code |
30332 |
| Country |
USA |
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| Platform ID |
GPL21697 |
| Series (1) |
| GSE116323 |
Bulk RNA-seq analysis of HUVEC cell cultures with Cerebral Cavernous Malformation (CCM) protein knockdown |
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| Relations |
| BioSample |
SAMN09499714 |
| SRA |
SRX4310928 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3227994_3D_2hrs_ccm3_vs_NT.diff.gz |
427.7 Kb |
(ftp)(http) |
DIFF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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