Molecular Cloning and Cell Line Generation

Human HNF4A and MYCN expression vectors were provided by Dr. David Martinez Selva [16 (link)] and Dr. Arturo Sala [17 (link)], respectively. Human HNF4A-AS1 cDNA (648 bp), hnRNPU cDNA (2478 bp), CTCF cDNA (2184 bp), and their truncations were amplified from NB tissues (Additional file 1: Table S2) and subcloned into pcDNA3.1 (Invitrogen), pCMV-3Tag-1C, pCMV-N-Myc, and pGEX-6P-1 (Addgene, Cambridge, MA), respectively. Mutation of short hairpin RNA (shRNA)-targeting site of HNF4A-AS1 or hnRNPU RGG residues was performed with GeneTailor^TM Site-Directed Mutagenesis System (Invitrogen) and primers (Additional file 1: Table S2). Oligonucleotides encoding shRNAs specific for HNF4A, HK2, SLC2A1, MYCN, HNF4A-AS1, hnRNPU, or CTCF (Additional file 1: Table S3) were subcloned into GV298 (Genechem Co., Ltd, Shanghai, China). Single guide RNAs (sgRNAs) targeting downstream region of HNF4A-AS1 transcription start site (Additional file 1: Table S3) were inserted into dCas9-BFP-KRAB (Addgene). Stable cell lines were screened by administration of neomycin or puromycin (Invitrogen).