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Sample GSM3227997 Query DataSets for GSM3227997
Status Public on Nov 19, 2018
Title 3D-CCM2-6hrs
Sample type SRA
 
Source name HUVEC_3D-CCM2
Organism Homo sapiens
Characteristics cell line: HUVEC
sirna for gene knockdown: ccm2 (LG-014728-01)
substrate for cultivation: Matrigel
time of incubation: 6 hours
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.
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.
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina MiniSeq
 
Description S11-3D-CCM2-6hrs_S1
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
 
Submission date Jun 27, 2018
Last update date Nov 20, 2018
Contact name Denis Tsygankov
E-mail(s) denis.tsygankov@bme.gatech.edu
Organization name Georgia Institute of Technology
Department Biomedical Engineering
Lab Integrative Systems Biology
Street address North Ave NW
City Atlanta
State/province GA
ZIP/Postal code 30332
Country USA
 
Platform ID GPL22790
Series (1)
GSE116323 Bulk RNA-seq analysis of HUVEC cell cultures with Cerebral Cavernous Malformation (CCM) protein knockdown
Relations
BioSample SAMN09499711
SRA SRX4310931

Supplementary file Size Download File type/resource
GSM3227997_3D_6hrs_ccm2_vs_NT.diff.gz 442.3 Kb (ftp)(http) DIFF
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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