293ltv cell line

SHSY5Y cells were purchased from ECACC (94030304) and cultured in DMEM containing 4 mM glutamine and 5.5 mM glucose, or 10 mM galactose, respectively. 293LTV cell line was purchased from Cell Biolabs, Inc. (LTV-100) and cultured in DMEM containing 4 mM glutamine and 5 mM glucose. The GCN5L plasmid was a kind gift from Dr. Sack’s laboratory^{19 (link)}. The IDH2 WT and IDH2 K413Q plasmids were a kind gift from the laboratory of Dr. Denu^{13 (link)}. Stable cell lines were generated by treatment with G418 (200 µg/ml) after transfection. SIRT3 and SIRT3 H248Y plasmids were purchased from Addgene (#24924, #24917 respectively) and respective ORFs subcloned into the p3XFLAG-CMV14 vector (Sigma-Aldrich). Vector DNA was transfected using Lipofectamine 2000 (ThermoFisher Scientific). For RNA silencing, the predesigned Mission siRNAs (Sigma-Aldrich) were transfected using Lipofectamine RNAiMAX (ThermoFisher Scientific). The decline of RNA was verified using real-time PCR and data calculated using Double Delta Ct analysis.

Six oligo duplexes targeting human GPR180 gene (Supplementary Tab. 3) were synthesized by Microsynth Inc. and inserted into plasmid lentiCRISPRv2 (Addgene, #52961) via BsmBI restriction site. lentiCRISPRv2-Gpr180 gRNA plasmids were transfected into 293LTV cell line (Cell Biolabs) together with pMD2.G (Addgene, #12259) and psPAX2 (Addgene, #12260) by PEI in Opti-MEM medium. The virus-containing medium was collected 2 days later and concentrated using PEG-it Virus Precipitation Solution (SBI, LV825A-1) according to the manufacturer’s instructions. The concentrated lentiviruses were added into medium to infect HEK-293T cells in presence of polybrene (Sigma, H9268). Two days later, the cells were suspended and selected by 1 µg/ml puromycin for 1 week to obtain a stable cell line. Afterwards, the HEK-293T cells were sorted into single cells using the Sony SH800S Cell Sorter (Sony Biotechnology) and cultured in 96-well plates for clonal expansion. Individual clones were validated using PCR and Sanger sequencing. Using this approach, we obtained a stable HEK-293T cell line with deletion of 83 bp (the sequence is available upon request) that led to a premature stop codon in exon 1 of GPR180 gene.