Maxi prep plasmid extraction kit

Coding sequences of OCT4, deleted mutants of OCT4, NRF1, FOXA1, and AR were amplified and cloned into expression vector pcDNA3.0 (Flag tagged or HA-tagged). Venus-Flag-tagged pcDNA3 was synthesized by amplifying Venus sequences from USE10C/pCS2 (RIKEN BRC DNA BANK) and subcloning them into pcDNA3.0 with Flag tag at C-terminal. Flag-mCherry-pcDNA3 was synthesized by amplifying mCherry sequences from pET-mCherry (RIKEN BRC DNA BANK) and subcloning into pcDNA3.0 with Flag tag at N-terminal. Then Venus-Flag-tagged AR, FOXA1, and NRF1 were synthesized by cloning coding sequences from cDNA of PC cells to EcoRV/XhoI restriction site. Flag- mCherry-tagged OCT4 was synthesized by subcloning OCT4 coding sequence to EcoRI/EcoRV restriction site. To construct luciferase vectors of OCT4BSs, each enhancer region was amplified by PCR and cloned into pGL3-SV40-promoter vector (Promega) using MluI/XhoI site. Plasmid DNA was transformed into competent cells for amplification and purified using Maxiprep Plasmid extraction kit (Qiagen).

Cells were transfected with 5 μg GFP-StarD13, dominant active RhoA, or control empty control vectors using Lipfectamine LTX with Plus reagent (Invitrogen) as described by the manufacturer. Cells were incubated with the transfection complexes for 4 h then refed with DMEM supplied with 30% FBS. The experiments were carried on 24 h following transfection. The GFP-StarD13 and the RhoA constructs were generous gifts from respectively Dr Hitoshi Yagisawa from the University of Hyogo, Japan and Dr Hideki Yamaguchi from the Albert Einstein College of Medicine, NY, USA.
The constructs were transformed into One Shot TOP10 chemically competent E. coli (Invitrogen), which were grown on a selective medium containing the appropriate antibiotic. The vectors were then extracted using MaxiPrep plasmid extraction kit from Qiagen. The mCherry-tagged RhoA-DA construct was a generous gift from Dr Louis Hodgson from Albert Einstein College of Medicine Yeshiva University, NY, USA.

Regulatory regions (R1 and R2), identified by integration of previously published human islet ATAC-seq and H3K27ac ChIP-seq datasets [11 (link)], were PCR-amplified from genomic DNA extracted from EndoC-βH3 cells with primer sets (Table S5) designed by Primer3-based software and cloned into pGL4.23[luc/minP] vector (Promega) between KpnI and XhoI restriction enzyme sites. Plasmid DNA was extracted using mini-prep plasmid extraction kit and/or Maxi-prep plasmid extraction kit (QIAGEN). The constructs were further confirmed by the Sanger sequencing.