ON-TARGETplus Human LIN28A (79727) siRNA - SMARTpool

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	The hESC lines H1 listed in the NIH hESC registry under the names of WA01 respectively) were obtained from WiCell. The iPSC line was generated at the Yale Stem Cell Center using Yamanaka factors (Takahashi et al. 2007). The cells were cultured in an undifferentiated state in Matrigel (BD Pharmingen CA)-coated plates under feeder-free and defined component conditions (Yao et al. 2006) at 37°C in hESC culture incubators (5% CO2, 5% O2, and 90%–95% humidity). Cell growth media were composed of Dulbecco's modified Eagle medium (DMEM)/F12 (Invitrogen), supplemented with 1% MEM-nonessential amino acids (Invitrogen), 1 mM L-glutamine, 1% penicillin-streptomycin (P/S), 50 ng/mL basic fibroblast growth factor (Millipore), N2 supplements (1×), and B27 supplements (1×) (Invitrogen). Cells were maintained with daily medium change and passaged weekly by dissociation into small clumps using 1 mg/mL dispase (StemCell Technology). The hESCs and iPSCs used were between passages 30 and 60 and 10 and 15, respectively. Free of mycoplasma contamination, the cells exhibited normal karyotype, expressed common ES markers, and were able to differentiate into the three germ layers (data not shown). siLin28 (ON-TARGETplus SMARTpool) and siCon were purchased from Dharmacon. To prepare siRNA/lipid solutions for each well of a six-well plate, 9 or 36 pmol of siRNAs were diluted in 50 μL of OPTI-MEMI (Invitrogen) and incubated at room temperature for 5 min. In a separate tube, 5 μL of Lipofectamine 2000 (Invitrogen) was diluted in 50 μL of OPTI-MEMI and incubation carried out for 5 min at room temperature. The contents of the two tubes were combined by gentle pipetting and incubated at room temperature for 30–50 min. The resulting 100 μL of transfection solution was used to resuspend the cell pellet (1 × 106) produced after Accutase dissociation (see above). For siRNA transfection using the standard method, cells were seeded in a 6-well plate 5 d prior to transfection. On the day of transfection, 100 μL of siRNA/lipid solution diluted in 1.5 mL of growth medium was added to each well of cells grown as single-layer colonies attached to the plate. ROCK inhibitors were not involved before, during, or after the transfection. RNAs were extracted for analysis 72 h after the transfection.

Protocol tips

The hESC lines H1 listed in the NIH hESC registry under the names of WA01 respectively) were obtained from WiCell. The iPSC line was generated at the Yale Stem Cell Center using Yamanaka factors (Takahashi et al. 2007). The cells were cultured in an undifferentiated state in Matrigel (BD Pharmingen CA)-coated plates under feeder-free and defined component conditions (Yao et al. 2006) at 37°C in hESC culture incubators (5% CO2, 5% O2, and 90%–95% humidity). Cell growth media were composed of Dulbecco's modified Eagle medium (DMEM)/F12 (Invitrogen), supplemented with 1% MEM-nonessential amino acids (Invitrogen), 1 mM L-glutamine, 1% penicillin-streptomycin (P/S), 50 ng/mL basic fibroblast growth factor (Millipore), N2 supplements (1×), and B27 supplements (1×) (Invitrogen). Cells were maintained with daily medium change and passaged weekly by dissociation into small clumps using 1 mg/mL dispase (StemCell Technology). The hESCs and iPSCs used were between passages 30 and 60 and 10 and 15, respectively. Free of mycoplasma contamination, the cells exhibited normal karyotype, expressed common ES markers, and were able to differentiate into the three germ layers (data not shown). siLin28 (ON-TARGETplus SMARTpool) and siCon were purchased from Dharmacon. To prepare siRNA/lipid solutions for each well of a six-well plate, 9 or 36 pmol of siRNAs were diluted in 50 μL of OPTI-MEMI (Invitrogen) and incubated at room temperature for 5 min. In a separate tube, 5 μL of Lipofectamine 2000 (Invitrogen) was diluted in 50 μL of OPTI-MEMI and incubation carried out for 5 min at room temperature. The contents of the two tubes were combined by gentle pipetting and incubated at room temperature for 30–50 min. The resulting 100 μL of transfection solution was used to resuspend the cell pellet (1 × 106) produced after Accutase dissociation (see above). For siRNA transfection using the standard method, cells were seeded in a 6-well plate 5 d prior to transfection. On the day of transfection, 100 μL of siRNA/lipid solution diluted in 1.5 mL of growth medium was added to each well of cells grown as single-layer colonies attached to the plate. ROCK inhibitors were not involved before, during, or after the transfection. RNAs were extracted for analysis 72 h after the transfection.

Publication protocol

The hESC lines H1 listed in the NIH hESC registry under the names of WA01 respectively) were obtained from WiCell. The iPSC line was generated at the Yale Stem Cell Center using Yamanaka factors (Takahashi et al. 2007). The cells were cultured in an undifferentiated state in Matrigel (BD Pharmingen CA)-coated plates under feeder-free and defined component conditions (Yao et al. 2006) at 37°C in hESC culture incubators (5% CO2, 5% O2, and 90%–95% humidity). Cell growth media were composed of Dulbecco's modified Eagle medium (DMEM)/F12 (Invitrogen), supplemented with 1% MEM-nonessential amino acids (Invitrogen), 1 mM L-glutamine, 1% penicillin-streptomycin (P/S), 50 ng/mL basic fibroblast growth factor (Millipore), N2 supplements (1×), and B27 supplements (1×) (Invitrogen). Cells were maintained with daily medium change and passaged weekly by dissociation into small clumps using 1 mg/mL dispase (StemCell Technology). The hESCs and iPSCs used were between passages 30 and 60 and 10 and 15, respectively. Free of mycoplasma contamination, the cells exhibited normal karyotype, expressed common ES markers, and were able to differentiate into the three germ layers (data not shown). siLin28 (ON-TARGETplus SMARTpool) and siCon were purchased from Dharmacon. To prepare siRNA/lipid solutions for each well of a six-well plate, 9 or 36 pmol of siRNAs were diluted in 50 μL of OPTI-MEMI (Invitrogen) and incubated at room temperature for 5 min. In a separate tube, 5 μL of Lipofectamine 2000 (Invitrogen) was diluted in 50 μL of OPTI-MEMI and incubation carried out for 5 min at room temperature. The contents of the two tubes were combined by gentle pipetting and incubated at room temperature for 30–50 min. The resulting 100 μL of transfection solution was used to resuspend the cell pellet (1 × 106) produced after Accutase dissociation (see above). For siRNA transfection using the standard method, cells were seeded in a 6-well plate 5 d prior to transfection. On the day of transfection, 100 μL of siRNA/lipid solution diluted in 1.5 mL of growth medium was added to each well of cells grown as single-layer colonies attached to the plate. ROCK inhibitors were not involved before, during, or after the transfection. RNAs were extracted for analysis 72 h after the transfection

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