Qiashredder column

TiHo-1403 and TiHo-0906 were isolated from the primary tumours of two animals included in this study (Fig. S1) and were established and cultured as previously described^{26 (link)}.Cultures with growth medium (medium 199 with 10% foetal bovine serum [FBS, Biochrom], and 2% penicillin–streptomycin) were incubated in a humidified atmosphere (5% CO₂) at 37 °C. To obtain pellets for RNA isolation, cells were detached using a dissociating agent (TrypLETM, Gibco). After centrifugation (1000 g for 10 min) of cell suspensions, the supernatant was discarded to obtain cell pellets. Cell pellets were resuspended in 1 mL freezing medium (medium 199 with 20% FBS, 2% penicillin–streptomycin and 10% DMSO [AppliChem]) and transferred into vials for cryogenic storage. Vials were preserved in a − 80 °C freezer during 24 h, and afterward, stored in a − 150 °C freezer until RNA extraction. For nucleic acids extraction, samples containing 4 × 10⁶ cells (TiHo-0906 p7 and p77, and TiHo-1403 p10) were homogenized using QIAshredderTM columns (QIAGEN). For all samples types (FFPE, FT and CL), RNA extraction was performed using the AllPrep DNA/RNA Mini Kit (QIAGEN) according to manufacturer’s instructions. RNA yields and purity (260/280 ratio) were measured with the Synergy 2 plate-reader (BioTek).

To analyze the gene expression of VEGFR-3 and Lyve-1 on the RNA level, total cellular RNA extraction was conducted using QIAshredder columns (Qiagen, Hilden, Germany) and an RNeasy Mini Kit (Qiagen, Hilden, Germany) as recommended by the manufacturer after DNase digestion. First, 750 ng of RNA was used for first-strand cDNA synthesis using the QuantiTect RT-Kit (Qiagen, Hilden, Germany). Measurements of the relative mRNA amounts of the target genes were conducted using the SYBRgreen dye technique on a LightCycler system (Roche Diagnostics, Mannheim, Germany). Ct values (cycle at which the SYBR Green intensity exceeds a threshold) were measured by using the LightCycler Software Version 4.05 (Roche). The relative gene expression rates were calculated using the 2^−ΔΔCt method [23 (link)] with normalization to the housekeeping gene β-actin.

Brains from adult zebrafish (cntn1b^−/−, caspr^−/−, mag^−/−) and 5dpf zebrafish larvae (nfascb^−/−) were snap-frozen and stored at −80 °C. For RNA isolation, samples were homogenized in RLT Buffer Plus (Qiagen) for 30 min on ice, followed by centrifugation in QIAshredder columns (Qiagen, 79654) for 2 min at maximum speed. RNA was isolated from lysates of whole brains or single larvae with the RNeasy Mini kit (Qiagen, 74104) and retrotranscribed with the SuperScript III First-Strand Synthesis system (ThermoFisher, 18080051). Quantitative real-time PCRs were performed with PowerUp SYBR Green Master Mix (ThermoFisher, A25742), using an Applied Biosystems 7500 Fast Real-Time PCR system. The following primers were used: cntn1b: 5’-TGGAAGAAATCGGCGACACA-3’ and 5’-TTCAGAAACGCAGGAGTGGT-3’, caspr: 5’-ATGGCAGACGGTTTTCCTCA-3’ and 5’-ACCTCCCACCTCCATGACTC-3’, nfascb: 5’-TAGACATCGTGACGCAGGGA-3’ and 5’-TATCCTCTCAAGAGACCTGTAATCA-3’, mag: 5’-TAGAGGAAGGCACGGGAGAC-3’ and 5’-GGGGCAGAGGGAATGATTGG-3’, Elf1a 5’-AGCAGCAGCTGAGGAGTGAT-3’ and 5’-GTGGTGGACTTTCCGGAGT-3’. Relative quantification of gene expression was performed by the ΔΔCt method.

RNA was prepared from stimulated PBMC stored at −70 °C in RLT buffer (Qiagen), using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and following the manufacturer’s instructions. Cells were homogenized with QIAshredder columns (Qiagen). After extraction, RNA was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific). RNA samples were stored at −70 °C. RNA was captured from stimulated CD4+ or CD8+ T cells stored at −20 °C in RNAlater (Thermo Fisher Scientific) after FACS sorting, according to a published protocol^{35 (link)}. Briefly, samples were mixed with lysis buffer (0.3% (v/v) Triton X100, 20 mM DTT, 2 mM dNTPs) in ratio 1:1 and vortexed. Magnetic Dynabeads (M-270 Streptavidin, Thermo Fisher Scientific) coated with RNA capturing polyT-tailed Indexing Oligonucleotides (Integrated DNA Technologies) were added to each well. After 5 min of incubation the magnetic beads were separated from the supernatant and washed twice with 6X SSC buffer. Subsequently, the beads were combined with RT mix as described later.