Inducible LEUTX cell lines used for NET-CAGE and ChIP-Seq were generated on hiPSC line HEL24.3. Inducible dCas9-activator cell line for endogenous gene activation was generated on hESC line H9 (WA09, WiCell).
HEL24.3. and H9 cells were treated with 10 μM ROCK inhibitor Y27632 (Selleckhem) for 4 h before electroporations. Cells were incubated with StemPro Accutase (Thermo Fisher Scientific) until the edges of the colonies started to curl up. The Accutase was aspirated and the cells were gently detached in cold 5% FBS (Thermo Fisher Scientific) 1×PBS (Corning) and counted. One million cells were centrifuged at 200xg for 5 min and the pellet was transferred into 120 μl of R-buffer containing 1 μg of either one of the LEUTX vectors (pB-tetON-LEUTX-ires-GFP-PGK-Puro/ pB-tetON-LEUTX-HA-V5-ires-GFP-PGK-Puro) or DDdCas9 plasmid cocktail below and 0.5 μg of transposase plasmid. 100 μl of the cell-plasmid suspension was electroporated with two pulses of 1100V, 20 ms pulse width, using Neon Transfection system (Thermo Fischer Scientific). Activator cell line was generated by electroporating H9 cells with two plasmids containing DDdCas9VP192 (1 μg of SB-tight-DDdCas9VP192- GFP-Zeo-WPRE) and rtTA (1 μg of SB-CAG-rtTA-IN-IRES-Neo) sequences, which were integrated into the genome by sleeping beauty transposase (0.5 μg of CAG-SB-100X-bghpA). Guide plasmids (1.5 μg / reaction) were electroporated into H9 DDdCas9VP192 activator cells and integrated with piggyBac transposase (0.5 μg of pCMV-HAhy-Pbase).
The electroporated cells were plated on Geltrex-coated dishes in Essential 8 medium with 10 μM ROCK inhibitor Y27632. The following day, the medium was exchanged with fresh Essential 8 medium without ROCK inhibitor. The cells were selected with Neomycin (G418, Life Technologies) at 50 μg/ml and Zeocin (Sigma) at 1 μg/ml (after DDdCas9VP192-GFP-Zeo-WPRE plasmid transfection) or Puromycin (Sigma) at 0.5 μg/ml (after LEUTX vectors and guide plasmids). The TetOn-LEUTX hPSC clones were picked manually on Geltrex-coated 24-well plates, expanded and selected again with Puromycin. Appearance of the GFP reporter protein was tested using Doxicycline at concentration 0.5 μg/ml and detected using an EVOS FL Cell imaging system (Thermo Fisher Scientific).
For the experiments presented in this paper, the LEUTX TetOn cells were treated with 1 μg/ml of Doxycycline for 6-7 h (NET-CAGE, q-PCR validation) or 24 h (ChIP-Seq, qPCR validation), DD-dCas9 activator cell line was treated with 1 μg/ml of Doxycycline and 1 μM Trimethoprim for 24 h, 48 h or 72 h, prior to harvesting cells for STRT-Seq.
HEL24.3. and H9 cells were treated with 10 μM ROCK inhibitor Y27632 (Selleckhem) for 4 h before electroporations. Cells were incubated with StemPro Accutase (Thermo Fisher Scientific) until the edges of the colonies started to curl up. The Accutase was aspirated and the cells were gently detached in cold 5% FBS (Thermo Fisher Scientific) 1×PBS (Corning) and counted. One million cells were centrifuged at 200xg for 5 min and the pellet was transferred into 120 μl of R-buffer containing 1 μg of either one of the LEUTX vectors (pB-tetON-LEUTX-ires-GFP-PGK-Puro/ pB-tetON-LEUTX-HA-V5-ires-GFP-PGK-Puro) or DDdCas9 plasmid cocktail below and 0.5 μg of transposase plasmid. 100 μl of the cell-plasmid suspension was electroporated with two pulses of 1100V, 20 ms pulse width, using Neon Transfection system (Thermo Fischer Scientific). Activator cell line was generated by electroporating H9 cells with two plasmids containing DDdCas9VP192 (1 μg of SB-tight-DDdCas9VP192- GFP-Zeo-WPRE) and rtTA (1 μg of SB-CAG-rtTA-IN-IRES-Neo) sequences, which were integrated into the genome by sleeping beauty transposase (0.5 μg of CAG-SB-100X-bghpA). Guide plasmids (1.5 μg / reaction) were electroporated into H9 DDdCas9VP192 activator cells and integrated with piggyBac transposase (0.5 μg of pCMV-HAhy-Pbase).
The electroporated cells were plated on Geltrex-coated dishes in Essential 8 medium with 10 μM ROCK inhibitor Y27632. The following day, the medium was exchanged with fresh Essential 8 medium without ROCK inhibitor. The cells were selected with Neomycin (G418, Life Technologies) at 50 μg/ml and Zeocin (Sigma) at 1 μg/ml (after DDdCas9VP192-GFP-Zeo-WPRE plasmid transfection) or Puromycin (Sigma) at 0.5 μg/ml (after LEUTX vectors and guide plasmids). The TetOn-LEUTX hPSC clones were picked manually on Geltrex-coated 24-well plates, expanded and selected again with Puromycin. Appearance of the GFP reporter protein was tested using Doxicycline at concentration 0.5 μg/ml and detected using an EVOS FL Cell imaging system (Thermo Fisher Scientific).
For the experiments presented in this paper, the LEUTX TetOn cells were treated with 1 μg/ml of Doxycycline for 6-7 h (NET-CAGE, q-PCR validation) or 24 h (ChIP-Seq, qPCR validation), DD-dCas9 activator cell line was treated with 1 μg/ml of Doxycycline and 1 μM Trimethoprim for 24 h, 48 h or 72 h, prior to harvesting cells for STRT-Seq.
Free full text: Click here
