(A) The transcriptional start site of CRED9 was detected in Hep3B cells by performing 5′ RLM-RACE. CRED9 –specific PCR products from the second round of nested PCR in lanes 8, 14, and 17 were generated from the same reverse transcription product (same gene-specific RT primer). The successful RT primer annealed +5022 nt downstream from the TSS of CRED9. Other lanes include unsuccessful amplification of CRED9 5′ RACE products resulting from RT primers: random hexamers (lane 2), oligo dT (lane 3), NoRT (lane 20), and gene-specific primers (all other lanes). (B) PCR amplified product from lanes 8, 14, and 17 were cloned into sequencing plasmids. Sequencing and subsequent alignment of the products showed different transcriptional start sites between Hep3b (yellow highlight), annotated transcript CATG00000039093.1 (blue highlight), and CAGE-seq data (red peaks). (C) Schematic of the CRED9 and the +9 kb CEBPA enhancer genomic region. 5′ RLM-RACE product (red), CATG00000039093.1 CAGE-seq transcript (blue), single guide RNAs (magenta), CRED9-specific antisense oligonucleotide gapmers (green). Primers used for 5′ RLM-RACE, droplet digital PCR, and ChIP-qPCR are also depicted.
