characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-06-01T00:15:24Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-06-01T00:15:24Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA111378962
characteristics.characteristic: External Id
characteristics.text: Queen Mary University of London; William Harvey Research Institute, Centre of Cardiovascular Medicine and Device
characteristics.characteristic: INSDC center name
characteristics.text: 2023-06-01T00:15:24Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-06-01T00:15:24Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS13475947
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12191:si-NRP1 Oscillatory 3
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: HUVEC cell line
characteristics.characteristic: cell_line
characteristics.text: endothelial cell of umbilical vein
characteristics.characteristic: cell_type
characteristics.text: human
characteristics.characteristic: common name
characteristics.text: Protocols: HUVECs transfected with siRNA targeting NRP1 or with a non-targeting siRNA were cultured in static conditions or exposed to laminar or oscillatory flow for 24 hours. RNAs from 3 independent experiments were isolated using the protocol described below and transcriptomic analysis performed by Novogene, UK. mRNA was collected using the Monarch® Total RNA Miniprep Kit (New England BioLabs, UK; cat#: T2010S) and cDNA was prepared using LunaScript® RT SuperMix Kit (New England BioLabs, UK; cat#: E3010L). A total amount of 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra TM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X) or by using sonication with Diagenode bioruptor Pico for breaking RNA strands. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.
characteristics.characteristic: description
characteristics.text: neonate
characteristics.characteristic: developmental stage
characteristics.text: normal
characteristics.characteristic: disease
characteristics.text: oscillatory flow
characteristics.characteristic: environmental stress
characteristics.text: not applicable
characteristics.characteristic: isolate
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: umbilical vein
characteristics.characteristic: organism part
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: si-NRP1 Oscillatory 3
characteristics.characteristic: title