Erase a base kit

Genomic DNA was isolated from a male C57BL/6N mouse liver using the Genomic DNA isolation kit (QIAGEN, Germantown, MD, USA). The CD24 promoter region from −688 to −1 from the TSS was amplified from genomic DNA with the GC-Rich PCR system (Roche, Basil, Switzerland) using the primers indicated in Table 1. The promoter was cloned into the HindIII and BglII sites of the pGL4.17 vector (Promega, Madison, USA). The deletion constructs −469 to −1, −357 to −1, and −168 to −1 were generated using the Erase-a-base kit (Promega) according to the manufacturer's instructions. All sequences were verified by sequencing at The Centre for Applied Genomics (Toronto, ON, Canada).
Control or RasV12 cells (3 × 10⁴ cells/well in 24-well-plates) were transfected with 1 μg of the pGL4.17 vector with or without the CD24 promoter regions and 6.66 ng pRL-SV40 vector (Promega) using 2.5 μl Superfect transfection reagent (Qiagen), following the manufacturer's instructions. After 24 h, cells were lysed with 1X Passive Lysis Buffer and Firefly and Renilla Luciferase activity were measured using the Dual-Luciferase Reporter Assay kit (Promega).

Plasmid isolation, restriction enzyme digests, agarose and polyacrylamide gel electrophoreses, electroporation, PCR, and other routine molecular methods were performed using standard protocols
[31 ]. Nested deletion analysis of the upstream region of lscB in plasmid pRB7.2
[10 (link)] was conducted using the Erase-a-Base kit (Promega, Madison, USA). For analysis of the lsc upstream regions, PCR was used to generate products covering the respective regions (Table 
3). PCR products of the lsc upstream regions were cloned in vectors pBBR1MCS or pBBR1MCS-3
[36 (link)].

Luciferase constructs were transfected into T47D using the GeneJuice transfection reagent (EMD Millipore, Darmstadt, Germany) according to manufacturer's recommendations. Constructs used include pGL3 Basic empty vector, Renilla expression construct, pGL3 containing TBXA2R 600 bp promoter construct and pGL3 vector containing shorter TBXA2R promoter constructs generated using the exonuclease mapping ERASE-A-BASE kit (Promega, Southampton, UK), according to manufacturer's directions. Luciferase activity was measured as previously described [24 (link)].