title: uninduced 1
organism: Mus musculus
description: Protocols: To collect samples for RNA sequencing, endothelial cells were cultured for 10 days after antibody sorting, before inducing TAZ-CAMTA1 expression or not with 1g/ml doxycycline. 24 hours later, endothelial cells were harvested using TrypLE Express enzyme (Gibco), then resuspended in IMDM 10% FCS and passed through a 50μm Filcon (Becton Dickinson). Endothelial cells were then sorted by flow cytometry into four populations; TAZ-CAMTA1 high, TAZ-CAMTA1 low, TAZ-CAMTA1-, and uninduced (dox-). TAZ-CAMTA1 expression levels were defined by GFP expression. The mouse embryonic stem cells (mESCs) contain a construct whereby TAZ-CAMTA1 expression is induced upon the addition of doxycycline. The TAZ-CAMTA1 sequence contains an N-terminal double FLAG epitope, and is followed by an IRES-GFP reporter cassette. In all experiments doxycycline was added to culture media at a final concentration of 1g/ml to induce protein expression. mESCs were expanded and maintained in DMEM-ES medium. This comprised of Dulbecco's Modified Eagle Medium (DMEM; Sigma) supplemented with 15% foetal calf serum (FCS), 2mM L-glutamine, 50 U/ml penicillin-streptomycin, 1% LIF conditioned media, and 0.15 mM MTG. Cells were maintained on a monolayer of mitomycin C inactivated mouse embryonic fibroblasts (MEFs) until differentiation. mESCs were cultured at 37C with 5% CO2 in a humidified incubator. To begin the differentiation, mESCs were passaged twice onto 0.1% gelatine coated plates without MEFs. The cells were first passaged in DMEM-ES, then in IMDM-ES. IMDM-ES contains IMDM supplemented with 15% foetal calf serum (FCS), 2mM L-glutamine, 50 U/ml penicillin-streptomycin, 1% LIF conditioned media, and 0.15 mM MTG. Next embryoid bodies (EBs) were generated by harvesting mESCs and transferring IMDM supplemented with 15% FCS, 2mM L-glutamine, 50 U/ml penicillin-streptomycin, 0.5 ng/ml ascorbic acid (Sigma), 180 µg/ml transferrin (Sigma), 0.45 mM MTG and 5ng/ml human vascular endothelial growth factor (VEGF; Peprotech). EBs were cultured in non-tissue culture treated 10cm dishes. From this point on, cells were maintained in low oxygen (5% O2) conditions, at 37C with 5% CO2 in a humidified incubator. At day 5 EBs were subject to cell sorting to isolate the TIE2+ FLK1+ population. After sorting, cells were cultured in endothelial cell medium, which contains IMDM with 10% FCS, 2mM L-glutamine, 50 U/ml penicillin-streptomycin, 180 µg/ml transferrin, 0.45 mM MTG, 0.25 ng/ml ascorbic acid, 15% D4T conditioned medium, 5ng/ml murine basic fibroblast growth factor (bFGF; Peprotech) and 5ng/ml human VEGF. Cells were maintained on Matrigel (Corning) diluted to a protein concentration of 5.5mg/ml in IMDM. Endothelial cell medium was replaced every 2-3 days and supplemented with doxycycline as appropriate. Cell pellets were resuspended in RLT lysis buffer from the RNeasy Mini Kit (Qiagen) and stored at -80C until sufficient samples for three biological repeats had been collected. RNA extraction was then performed on all samples at the same time, using the RNeasy Mini Kit, as per manufacturer's instructions. RNA concentration was quantified using the Qubit RNA BR Assay Kit (Invitrogen) and Qubit 3 fluorometer (Invitrogen). Illumina TruSeq Stranded mRNA kit, according to the manufacturer's protocol.