Pcpgfree promoter lucia plasmid

Promoter luciferase assays were performed by cloning chr6:18122895-18123314 (Supplementary Table S2 and Figure S6) into the pCpGfree-promoter-LUCIA plasmid (Invivogen, San Diego, CA, USA). In vitro M.SssI (Thermo Scientific) methylated or unmethylated pCpGfree-chr6:18122895-18123314 plasmids were co-transfected with pGL4-SV40-promoter (Promega, Madison, WI, USA) plasmid [59 (link)] into A549 and H1299 with the transfection reagent Lipofectamine 3000 (Thermo Scientific). Chemiluminescence was measured in a Tecan200pro plate reader (Tecan) in technical quadruplicates. Renilla readouts were normalized to Firefly luciferase and empty pCpGfree vector readouts.

A 568bp region encompassing the 14 CpGs was amplified using genomic DNA (primer sequences in Supplementary Table 2), then cloned into the pCR-Blunt-II-TOPO vector (Invitrogen) and transformed into chemically competent E. coli. Colonies were grown overnight; plasmid DNA was extracted, and Sanger sequenced (Source Bioscience). Plasmids containing the desired insert were digested using the restriction enzymes AvrII and SpeI (New England Biolabs). The insert was cloned into a pCpGfree-promoter-Lucia plasmid (InvivoGen), then methylated and mock-methylated using M.SssI (New England Biolabs). Tc28a2 immortalised chondrocytes [22 (link)] were seeded onto a 96-well plate at 5000 cells/well and transfected 24h later with 100ng pCpG-free-promoter DNA and 10ng pGL3-promoter (Promega) with Lipofectamine 2000 (Invitrogen). Cells were lysed after 24h and luminescence was read using the Dual-Luciferase Reporter Assay System (Promega) [15 (link)]. Six biological replicates were performed for each plasmid construct.

A 282 bp region encompassing the CpGs was amplified using genomic DNA and the insert was cloned into the pCpG-free-Basic-Lucia plasmid (Invivogen) to assess promoter function, and into the pCpG-free-Promoter-Lucia plasmid (Invivogen) to assess enhancer function (primer sequences available in Additional file 3). Successful cloning was determined by Sanger sequencing (Source Bioscience). Plasmids were methylated or mock-methylated using M.SssI (New England Biolabs). Tc28a2 immortalised chondrocytes [40 (link)] were seeded at a density of 5000 cells/well in 96-well plates and incubated for 24 h. Then, cells were transfected with 100ng insert DNA-vector constructs and 10ng pGL3-promoter-luciferase plasmid (Promega) using Lipofectamine 2000 (Invitrogen) and incubated. After 24 h, cells were lysed, and luminescence measured using the Dual Luciferase Reporter Assay system (Promega). Twelve biological replicates were performed per plasmid.