Lentiviral transduction for stable cell lines

Lentiviral vectors expressing YTHDF2 and W432A/W486A/W491A mutants were PCR amplified and cloned into pLVX-puro vector (Clontech). Lentiviral vectors expressing non-targeting pLKO.1 control shRNA (SCH002), and shRNA constructs targeting YTHDF2 ({"type":"entrez-nucleotide","attrs":{"text":"NM_016258","term_id":"1519315892","term_text":"NM_016258"}}NM_016258) shRNA#1 (TRCN0000254410) and shRNA#2 (TRCN0000265510), or targeting LXRA ({"type":"entrez-nucleotide","attrs":{"text":"NM_005693","term_id":"1519245877","term_text":"NM_005693"}}NM_005693) shRNA#1 (TRCN0000022238) and shRNA#2 (TRCN0000438551), or targeting HIVEP2 ({"type":"entrez-nucleotide","attrs":{"text":"NM_006734","term_id":"1519242967","term_text":"NM_006734"}}NM_006734) shRNA#1 (TRCN0000236545) and shRNA#2 (TRCN0000022097) were obtained from Sigma-Aldrich. shRNA for LXRA and HIVEP2 were generated according to the pLKO.1 protocol from Addgene. The lentiviral vectors were co-transfected with packaging vectors psPAX2 and pMD2.G (Addgene) into 293FT cells for lentivirus production. To establish stable cell lines, GSC cells or LN229 cells were transduced by the above lentiviruses with polybrene (8 µg/mL, Sigma-Aldrich). After 72 h of transduction, GSC cells were selected with 2 µg/mL puromycin, 5 µg/mL blasticidin, or 400 µg/mL geneticin (G418), and LN229 cells were selected with 0.5 µg/mL puromycin for 1 week and expanded as polyclonal populations. For the YTHDF2 rescue experiment, shRNA targeting 3′UTR of YTHDF2 (shYTHDF2#1) was used for knockdown.