Plvx ef1α ires puro

A construct overexpressing FGF14-AS2 was generated by ligating the full-length human FGF14-AS2 into vector pLVX-EF1α-IRES-puro (Clontech, Mountain View, CA, USA). For FGF14-AS2 knockdown, shRNA oligos against FGF14-AS2 were synthesized, annealed, and ligated into vector pLentilox 3.7 (Addgene, Cambridge, MA, USA), with a nontargeting control sequence (shNC) as the control. Table S3 lists the primers used. High-titer lentivirus was packaged in HEK 293T cells. The viral particles were collected by centrifugation at 48 h post transfection, and applied to cells in the presence of 5 μg/mL polybrene for 48 h. FGF14-AS2-overexpressing cells were selected using puromycin (5 μg/mL) for 2 weeks. Gene knockdown and overexpression were confirmed by qRT-PCR.

Dual-luciferase reporter vector psiCHECK-2 (Promega) carrying either the wild-type (WT) E2F motifs or a mutant version (Mut), and the vector pLVX-EF1α-IRES-puro (Clontech) carrying FLAG-tagged SAPCD2 were constructed by General Biosystems (Anhui, China). pLVX-EF1α-IRES-puro carrying HA-tagged E2F7 was constructed by Tsingke Biological Technology (Beijing, China). Recombinant constructs were verified by Sanger sequencing. Cells were transfected using Lipofectamine 3000 (Thermo Fisher Scientific). After 48 h of transfection, the luciferase activity was measured by the Dual-Luciferase Reporter Assay System (Promega).

Seventy to ninety percent confluent Lenti-X 293T HEK cells cultured in DMEM 10% FBS media were transfected with pMD2.G and pCMVR8.74 alongside appropriate delivery plasmids: pLV[Exp]-U6 > sgRNA-hPGK > mApple (Vectorbuilder) plasmids (for sgRNA), empty pLVX-EF1α-IRES-Puro (Clontech, Takara Bio, 631988), V5-KANSL1 pLVX-EF1α-IRES-Puro or V5-KAT8 pLV[Exp]-EF1α-IRES-Puro at a 1:1:2 molar mass ratio using Lipofectamine 3000 (Invitrogen). The next day, a full media change was performed with mTeSR1 (for sgRNA lentivirus) or DMEM 10% FBS media and cells cultured for 24 h. The lentivirus containing mTeSR1/DMEM 10% FBS was collected and diluted 1:2 with fresh mTeSR1 or 10% FBS before filtering through 0.44 µm PES filters. pMD2.G (Addgene plasmid no. 12259, RRID:Addgene_12259) and pCMVR8.74 (Addgene plasmid #22036, RRID:Addgene_22036) were gifts from Didier Trono. KANSL1 cDNA (ENST00000432791.7) with N-terminal V5 tag was cloned into the pLVX-EF1α-IRES-Puro plasmid using SpeI and NotI restriction sites (doi:10.5281/zenodo.6903553). See Supplementary Table 1 for sgRNA plasmids and V5-KAT8 pLV[Exp]-EF1α-IRES-Puro plasmids (Vectorbuilder).

To generate a luciferase-expressing lentiviral plasmid, the plasmid pGL4.10[luc2] (Promega) was used as PCR template to amplify the luciferase (luc2) gene. The PCR product was cloned into the lentiviral plasmid pLVX-EF1α-IRES-puro (Clontech). Luciferase-expressing lentiviral vectors were then generated by calcium phosphate co-precipitation. Molm14 cells were transduced with the pLV-luc2-puro vectors at an MOI of 0.5 and selected with 1 μg/ml puromycin (Sigma).

LentiCas9-Blast was a gift from Feng Zhang (Addgene plasmid # 52962; http://addgene.org/52962; RRID:Addgene_52962). The plasmid psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260; htto://addgene.org/12260; RRID:Addgene_12260). The plasmid pLenti-sgRNA was a gift from Eric Lander and David Sabatini (Addgene plasmid # 71409; http://addgene.org/71409; RRID:Addgene_71409). The plasmid pHEF-VSVG was a gift from Sergey Kasparov (Addgene plasmid # 22501; http://addgene.org/22501; RRID:Addgene_22501). The HLA expression set included A^∗0101, A^∗0201, A^∗0301, A^∗2402, B^∗0702, B^∗0801, B^∗1402, B^∗1501, B^∗2705, B^∗3501, B^∗3901, B^∗4001, B^∗4402, B^∗5201, B^∗5701, B^∗5801, B^∗8101 and Cw^∗0701. Synthetic HLA allele fragments (LifeSct) were cloned into a modified pLVX-EF1α-IRES-Puro (Clontech) vector (46), in which EF1α was replaced with the SFFV promoter (pLVX-SFFV-IRES-Puro). This expression cassette also encoded ZsGreen linked via self-cleaved P2A peptide to HLA with a FLAG-tag at its N terminus. These elements were removed by enzymatic digestion with EcoRI (NEB) and NotI (NEB) prior to re-cloning of HLA fragments. TAP sgRNA construct (5′-CACCGCGGGATCTATAACAACACCA-3′) was cloned into pLenti-sgRNA (McKinley et al., 2015 (link); Park et al., 2017 (link)). All plasmids were confirmed by complete plasmid sequencing (MGH DNA Core).

The HOTAIR expression construct LZRS-HOTAIR was kindly provided by dr. Howard Chang, Stanford University, USA, and the HOTAIR coding region was subcloned into the retroviral vector pLVX-EF1α-IRES-Puro (Clontech). HOTAIR shRNA vectors (GV248, HOTAIR-shRNA 1, 2, 3 and control shRNA) were purchased from GeneChem (Shanghai, China). Transfection of plasmids was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Stable shRNA expressing colonies were selected using puromycin.