Dnase 1 treatment

After MAEC culture with morpholino oligomer, total RNA was purified with an RNeasy mini kit (Qiagen, Germantown, MD), followed by DNaseI treatment (Sigma-Aldrich, St. Louis, MO). cDNA was prepared with an RT Omniscript kit (Qiagen) using oligo-dT. Real-time polymerase chain reaction (PCR) was carried out with SYBR green PCR (Qiagen) using previously described primers and PCR reaction conditions.^{12 (link),13 (link)} Real-time data were analyzed with the ΔΔCt method. sFLT protein quantification from the conditioned culture medium and mouse serum was performed by Mouse sVEGF R1/Flt-1 ELISA (R&D Systems, Inc., Minneapolis, MN) following the manufacture's instruction.

Cells were resuspended in 1 ml of non-denaturing cell lysis buffer (20 mM Tris–HCl pH 8, 137 mM NaCl, 1% IGEPAL CA 630, 2 mM EDTA, 2× protease inhibitor, RNase inhibitor) and passed through a 19-gauge needle to sheer the DNA. The samples were incubated with over-end rotation at 4°C for 30 min followed by centrifugation to remove the cell debris. Five percent of each lysate was stored for later to be used as input samples. In the immunoprecipitation, 50 μl of protein G Dynabeads (Thermo Scientific) and 1 μg of antibody were incubated with 500 μl of pre-cleared cell lysate with over-end rotation at 4°C for 2 h. Antibodies used are listed in Supplementary Table S4. Following incubation, the beads were washed eight times using wash buffer (20 mM Tris–HCl pH 8, 137 mM NaCl, 1% IGEPAL CA 630, 2 mM EDTA) and RNA was extracted using using Trizol-chloroform, followed by DNase I treatment (Sigma), purified by phenol–chloroform–IAA and precipitated by ethanol. The precipitated RNA was resuspended in 22 μl of ddH₂O and 10 μl was reverse transcribed using M-MLV reverse transcriptase (Invitrogen) and random hexamer primers (Thermo Scientific) according to the manufacturer's protocol. A 1 μl aliquot of cDNA was subjected to PCR amplification using the primers indicated in each figure.