Lipofectamine 2000 l2k

Concentrations of IFNβ were determined with the VeriKine-HS human IFNβ serum ELISA kit (PBL Assay Science, Piscataway, NJ, USA). TNF and IL-6 ELISA duo-kits were from R&D Systems, and BioPlex assays were from Bio-Rad. The cytokine levels were determined as per the manufacturer’s instructions. The PRR ligands FSL1, CL75, LPS O111:B4, poly(deoxyadenylic-deoxythymidylic) acid (poly-dA:dT; B-DNA), polyinosinic-polycytidylic acid [poly(I:C)], and polyuridylic acid (pU) were purchased from Inviviogen. Poly-l-arginine (pLA) was from Sigma. Lipofectamine 2000 (L2K) was purchased from Invitrogen.

Guide strands were designed using public resources (http://crispr.mit.edu) (see Supplementary Table S1 for detailed oligonucleotide sequences), and were cloned into px330 SV40-GFP vector (gift from Dr Eduard Batlle, IRB Barcelona) (43 (link)) as described in (44 (link)).
HEK293T cells growing in six-well plate format were transfected with 3 μg px330 SV40-GFP (CTRL), px330 SV40-GFP ADAT2 (ADAT2 KD) or px330 SV40-GFP ADAT3 (ADAT3 KD) constructs using lipofectamine 2000 (L2K) (11668027, Thermo Fisher) following the manufacturer's protocol (250 μl plasmid/lipid reaction in 2 ml DMEM Full Media). Forty-eight hours later, GFP-positive cells were sorted using a FacsAria I SORP sorter (Beckton Sickinson). Sorting on 96-well plates was done using an ACDU system, and one cell per well was sorted in wells containing 100 μl DMEM Full Media (see Supplementary methods). Out of 96 clones analysed per cell line, 55 and 74 were inviable when they were derived from px330 SV40-GFP ADAT2 or px330 SV40-GFP ADAT3 treated cells, respectively. Out of the viable clones, none presented a full KO of the targeted gene, suggesting that full ablation of ADAT2 or ADAT3 is lethal in this cell line. 100% of the single cell seeded clones derived from px330 SV40-GFP treated cells (CTRL) were viable. DNA edition was confirmed by sequencing.

1,2‐di‐O‐octadecenyl‐3‐trimethylammonium propane (DOTMA), 1,2‐dioleoyl‐sn‐glycero‐3‐phospho‐(1′‐rac‐glycerol) (DOPG), 1,2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine (DOPE), and 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐[methoxy(polyethylene glycol)‐2000] (DPPE‐PEG₂₀₀₀) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Integrin‐targeting peptide ME27 (K₁₆RVRRGACRGDCLG) and non‐targeting control peptide ME72 (K₁₆RVRRGACRGECLG) were synthesized by China Peptides (Shanghai, China).
siMYCNs for in vitro transfections were purchased from Eurogentec (Seraing, Liege, Belgium) with sense strands shown below;
MYCN‐1: 5′‐CGGAGAUGCUGCUUGAGAA‐3′
MYCN‐2: 5′‐CGGAGUUGGUAAAGAAUGA‐3′
MYCN‐3: 5′‐CAGCAGUUGCUAAAGAAAA‐3
The in vivo siRNAs, including MYCN‐3 and control siRNA (UGGUUUACAUGUUGUGUGA) were purchased from GE Healthcare (Amersham, UK) while Silencer Negative Control #1 (Irrelevant control siRNA 5′‐UAACGACGCGACGAACGUAATT‐3′) was purchased from Applied Biosystems (Warrington, UK). Labeled siRNAs including control siRNA‐fluorescein (siRNA‐FAM) and Dy677 control siRNA (siRNA‐Dy677) were purchased from GE Healthcare (Amersham, UK).
Lipofectamine 2000 (L2K) was purchased from Thermo Fisher (Paisley, UK) and mouse serum from Sigma‐Aldrich (Gillingham, UK).