0.45 μm syringe filter

Snap-frozen cecal content was thawed on ice and weighed (30 mg/sample) and homogenized in 600 μl of 25% phosphoric acid. Samples were centrifuged at 15,000 rpm at 4 °C for 10 min, and the supernatant was passed through a 0.45 μm syringe filter (Fisher). A 200 μl aliquot of filtered sample was combined with 50 μl of internal standard (23 μmol/ml, isocaproic acid) and analyzed on a SCION 456-GC instrument.

Lentiviral production and infection were carried out by co-transfecting various complementary DNA (cDNA) and packaging plasmids into 293T cells using Lipofectamine 2000 reagent according to manufacturer's instruction (#52758, Invitrogen). Forty-eight hours after transfection, the supernatants containing viruses were filtered by a 0.45-μm syringe filter (Fisher) and added into the culture media supplemented with 8 μg ml⁻¹ polybrene. Forty-eight hours after the infection, transduced glioma spheres were collected and re-cultured in stem cell media. Cells with GFP expression were sorted using fluorescence-activated cell sorting (FACS). Expression of exogenous proteins or effects of shRNA knockdowns on targeting protein expression in the resultant cell populations was validated by IB and GFP expression by FACS55 (link).

We extracted 5 g of pulp, peel, and the seed of the three avocado varieties with 20 mL of 80% (v/v) ethanol and homogenised for 30 s with the Ultra-Turrax T25 Homogenizer (Jane & Kunkle IKA-Labortechnik, USA). Then, all samples were incubated at 120 rpm at 4 °C in a shaking incubator (ZWYR-240, Labwit, Ashwood, VIC, Australia) for 12 h. Then, samples were centrifuged at 5000 rpm at 4 °C for 15 min in a benchtop centrifuge (Zentrifugen Rotina 380R, Hettich, Germany). Then, the supernatant was collected and filtrated through 0.45 μm syringe filter (Thermo Fisher Scientific Inc., Waltham, MA, USA) for further analysis.

PCR amplification of mouse Robo4 was performed with primers incorporating Age 1 (CCGG) and EcoR 1 (AATTCAAAAA) restriction sites (Invitrogen, Carlsbad, USA). The synthesized mRobo4 oligonucleotides: 5′-GCTGACTGTGTCTTCACTGAT CTCGAG ATCAGTGAAGACACAGTCAGC-3′ (Robo4-KD#1), 5′-GCCAACAACCTATGGCTATAT CTCGAG ATATAGCCATAGGTTGTTGGC-3′ (Robo4-KD#2) and 5′-GCCACCAACAATGCTGGGCAA CTCGAG TTGCCCAGCATTGTTGGTGGC-3′ (Robo4-KD#3) with or without the 3′-UTR (TTTTTG) were inserted and cloned into GV248 vector (hU6-MCS-Ubiquitin-EGFP-IRES-puromycin), (Genechem, Shanghai, China) according to Genechem standard procedures and validated by restriction digestion and DNA sequencing. As a transfection control (CON.), we utilized a plasmid bearing a control sequence that did not target any particular gene (TTCTCCGAACGTGT CACGT). Also, Genechem supplied lentiviral vectors harboring Robo4 overexpression vectors (Robo4-OE) and its control vector (CONT). The packaging cell line HEK293T cells were co-transfected with the recombinant GV248 vector plasmid (20 μg) containing the shRobo4 and Robo4-OE vectors, 15 μg pHelper 1.0 vector plasmid (pGAG-POL), and 10 μg pHelper 2.0 vector plasmid (pCMV-VSVG). Two days after transfection, viral supernatant from 293 T cells was filtered through a 0.45 μm syringe filter (Thermo Fisher) and ultracentrifuged (Beckman).