Xfect mesc transfection reagent

pCMV-dCAS9-VP64 vector (C9V64) and sgCdx2Ps vector were co-transfected into OG2 mESCs using Xfect™ mESC Transfection Reagent (Clontech) as mentioned above. pcDNA3.1 and pCMV-Cdx2 vector were used as negative and positive control, respectively. The following day, the ESCs were dissociated into single cells and seeded to 1 × 104 (link) on 35 mm tissue culture dishes with feeder layer. After another day, the culture medium was refreshed with TSC-1640 medium, and performed every other day. After TSC colony formation, the medium was changed to TSC-X medium. Endogenous Gata6 was activated the same way with XEN medium replaced 1 day after transfection.

The immunostaining on testis cryosections is as described [54 (link)]. Ezh2KO and EzhDKO ES cells were transfected with FLAG-tagged EZH variants using Xfect™ mESC Transfection Reagent (631320, Clontech) according to manufacturer’s instruction. The cells were trypsinized and resuspended in ES cell medium 48 h post-transfection. ES cells were fixed on slides by adding three volumes of 2% PFA (containing 0.1% Triton X-100 and 100 mM sucrose in 1 × PBS) at 4 °C for 30 min. The slides were air-dried and stored at 4 °C for immunofluorescence assays.

Wild-type and Dnmt2 KO MEF cells were prepared as described [13 (link)] and were grown in standard Dulbecco's modified Eagle's media containing 10% fetal bovine serum and supplemented with 2 mm Glutamate in a 5% CO₂ incubator. The cells were grown to 90% confluence and split into a six-well plate prepared with cover slides at a seeding density of 1×10⁶ cells per well. Afterwards, the cells were grown for another 24 h in Dulbecco's modified Eagle's medium to reach ~50% confluence. Then, the MEF cells were transfected with a fluorescent reporter construct (yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)) with and without poly-Asp₆-coding sequence at their N-terminal side using Xfect mESC Transfection Reagent (Clontech, Mountain View, CA, USA) following the instructions of the supplier. The medium was exchanged 3 h after transfection.

U6-sgCdx2-hCAS9 and donor vectors in ratio of 1:3 (w/w) were transfected into OG2 ESCs using Xfect™ mESC Transfection Reagent (Clontech) as instructed. U6-sgCdx2-hCAS9 contained a Neomycin gene (neo), conferring G418 resistant ability to the transfected cells. ESC medium was refreshed everyday with 800 μg/mL G418 (Sigma) for one week. G418 resistant colonies were retrieved for further culture. Portion of each colonies was collected for genomic PCR to detect 2A-td Tomato insertion. The primers are ROPG-F: 5′-TTGATTTATGGAAAGGAGGGGTGC-3′ and ROPG-R: 5′-GTGGAGGTGGGAGTGGGAAGATAATG-3′.