alias: GSM3227992
title: GSM3227992: 3D-CCM1-2hrs; Homo sapiens; RNA-Seq
library_construction_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.
identifiers.namespace: GEO
identifiers.id: GSM3227992