siRNA / miRNA gene silencing Human - hES cell line H1 (WA01) B2M

miRNA is the inherent gene silencing machinery which can have more than one mRNA target, whereas siRNA can be designed to target a particular mRNA target. By design, both siRNA and miRNA are 20-25 nucleotides in length. The target sequence for siRNAs is usually located within the open reading frame, between 50 and 100 nucleotides downstream of the start codon. There are two ways in which cells can be transfected with desired RNAi: 1. Direct transfection (with calcium phosphate co-precipitation or cationic lipid mediated transfection using lipofectamine or oligofectamine), and 2. Making RNAi lentiviral constructs (followed by transformation and transduction). Lentiviral constructs are time consuming, but provide a more permanent expression of RNAi in the cells, and consistent gene silencing. Direct transfection of oligonucleotides provides temporary genetic suppression. Traditional methods like calcium phosphate co-precipitation have challenges like low efficiency, poor reproducibility and cell toxicity. Whereas, cationic lipid-based transfection reagents are able to overcome these challenges, along with applicability to a large variety of eukaryotic cell lines. When using oligos, the ideal concentration lies between 10-50nM for effective transfection.

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B2M siRNA

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	hES cell line H1 (WA01, both National Stem Cell Bank) was cultured on Matrigel (BD Biosciences)-coated 6-well tissue culture plates (Corning) in mTeSR1 medium (STEMCELL Technologies) which was changed daily. Cells were mechanically passaged every 3–4 days using micropipette tip for detaching and breaking the colonies into pieces followed by plating onto fresh Matrigel-coated plates. Prior to transfection cells were passaged with EDTA-PBS to achieve a suspension of small cell clumps (described below). Cells were cultured at 37 °C in 5% CO2 and in a humidified atmosphere. hES cells were washed and incubated in 0.5 mM EDTA in phosphate-buffered saline (PBS). The plate was incubated on the warm surface (37 °C) for 8–10 min. Cell suspension was obtained by slow pipetting with micropipette for several times, and it was transferred to a 15-ml tube followed by centrifugation at 200 g for 5 min. Supernatant was removed, and cells were resuspended in fresh medium and counted by Countess II (ThermoFisher). Cell suspension with suitable concentration of cells was prepared, and 1 ml of suspension was added to each well. Three different plating densities were used, namely 3 × 105 or 4.5 × 105 or 6 × 105 cells per well of 6-well plate. Silencer® select pre-designed B2M siRNA (siB2M, ID: s1854), and Negative control #1 siRNA (siCtrl) were obtained from Ambion (ThermoFisher). PepFect 14 (PF14, sequence: stearyl-AGYLLGKLLOOLAAAALOOLL-NH2) was purchased from Pepscan. PF14 1-mM aliquots were stored at − 20 °C. For transfection, aliquots were diluted to 100 μM with MQ water and used for maximum 3 independent experiments (i.e., maximum storage of 3 weeks at 4 °C).On the day of experiment, 20-μM aliquots of siRNA in water were thawed and diluted in MQ water followed by addition of PF14 and mixing by pipetting. Complexes were formed in 200 μl (1/10th of final transfection volume) for 1 h at RT.siRNA and PF14 concentrations were adjusted proportionally to achieve nanocomplexes at charge ratio (CR, also known as N/P) of 2:1 and molar ratio (MR) of 16:1 (peptide:siRNA). For controlled complex formation, a solution for 30 nM siRNA final concentration was prepared and the volume of solution added to the cells varied accordingly. Transfection complexes of Lipofectamine Stem reagent (ThermoFisher) and siRNA (final concentration of 20 nM) were prepared according to manufacturer’s instructions. siOCT4 or siB2M was transfected for 24 h, and medium was changed as shown in Fig. 2a. Cells were analyzed at 48 h or 72 h.

Protocol tips
hES cell line H1 (WA01, both National Stem Cell Bank) was cultured on Matrigel (BD Biosciences)-coated 6-well tissue culture plates (Corning) in mTeSR1 medium (STEMCELL Technologies) which was changed daily. Cells were mechanically passaged every 3–4 days using micropipette tip for detaching and breaking the colonies into pieces followed by plating onto fresh Matrigel-coated plates. Prior to transfection cells were passaged with EDTA-PBS to achieve a suspension of small cell clumps (described below). Cells were cultured at 37 °C in 5% CO2 and in a humidified atmosphere. hES cells were washed and incubated in 0.5 mM EDTA in phosphate-buffered saline (PBS). The plate was incubated on the warm surface (37 °C) for 8–10 min. Cell suspension was obtained by slow pipetting with micropipette for several times, and it was transferred to a 15-ml tube followed by centrifugation at 200 g for 5 min. Supernatant was removed, and cells were resuspended in fresh medium and counted by Countess II (ThermoFisher). Cell suspension with suitable concentration of cells was prepared, and 1 ml of suspension was added to each well. Three different plating densities were used, namely 3 × 105 or 4.5 × 105 or 6 × 105 cells per well of 6-well plate. Silencer® select pre-designed B2M siRNA (siB2M, ID: s1854), and Negative control #1 siRNA (siCtrl) were obtained from Ambion (ThermoFisher). PepFect 14 (PF14, sequence: stearyl-AGYLLGKLLOOLAAAALOOLL-NH2) was purchased from Pepscan. PF14 1-mM aliquots were stored at − 20 °C. For transfection, aliquots were diluted to 100 μM with MQ water and used for maximum 3 independent experiments (i.e., maximum storage of 3 weeks at 4 °C).On the day of experiment, 20-μM aliquots of siRNA in water were thawed and diluted in MQ water followed by addition of PF14 and mixing by pipetting. Complexes were formed in 200 μl (1/10th of final transfection volume) for 1 h at RT.siRNA and PF14 concentrations were adjusted proportionally to achieve nanocomplexes at charge ratio (CR, also known as N/P) of 2:1 and molar ratio (MR) of 16:1 (peptide:siRNA). For controlled complex formation, a solution for 30 nM siRNA final concentration was prepared and the volume of solution added to the cells varied accordingly. Transfection complexes of Lipofectamine Stem reagent (ThermoFisher) and siRNA (final concentration of 20 nM) were prepared according to manufacturer’s instructions. siOCT4 or siB2M was transfected for 24 h, and medium was changed as shown in Fig. 2a. Cells were analyzed at 48 h or 72 h.

Protocol tips

hES cell line H1 (WA01, both National Stem Cell Bank) was cultured on Matrigel (BD Biosciences)-coated 6-well tissue culture plates (Corning) in mTeSR1 medium (STEMCELL Technologies) which was changed daily. Cells were mechanically passaged every 3–4 days using micropipette tip for detaching and breaking the colonies into pieces followed by plating onto fresh Matrigel-coated plates. Prior to transfection cells were passaged with EDTA-PBS to achieve a suspension of small cell clumps (described below). Cells were cultured at 37 °C in 5% CO2 and in a humidified atmosphere. hES cells were washed and incubated in 0.5 mM EDTA in phosphate-buffered saline (PBS). The plate was incubated on the warm surface (37 °C) for 8–10 min. Cell suspension was obtained by slow pipetting with micropipette for several times, and it was transferred to a 15-ml tube followed by centrifugation at 200 g for 5 min. Supernatant was removed, and cells were resuspended in fresh medium and counted by Countess II (ThermoFisher). Cell suspension with suitable concentration of cells was prepared, and 1 ml of suspension was added to each well. Three different plating densities were used, namely 3 × 105 or 4.5 × 105 or 6 × 105 cells per well of 6-well plate. Silencer® select pre-designed B2M siRNA (siB2M, ID: s1854), and Negative control #1 siRNA (siCtrl) were obtained from Ambion (ThermoFisher). PepFect 14 (PF14, sequence: stearyl-AGYLLGKLLOOLAAAALOOLL-NH2) was purchased from Pepscan. PF14 1-mM aliquots were stored at − 20 °C. For transfection, aliquots were diluted to 100 μM with MQ water and used for maximum 3 independent experiments (i.e., maximum storage of 3 weeks at 4 °C).On the day of experiment, 20-μM aliquots of siRNA in water were thawed and diluted in MQ water followed by addition of PF14 and mixing by pipetting. Complexes were formed in 200 μl (1/10th of final transfection volume) for 1 h at RT.siRNA and PF14 concentrations were adjusted proportionally to achieve nanocomplexes at charge ratio (CR, also known as N/P) of 2:1 and molar ratio (MR) of 16:1 (peptide:siRNA). For controlled complex formation, a solution for 30 nM siRNA final concentration was prepared and the volume of solution added to the cells varied accordingly. Transfection complexes of Lipofectamine Stem reagent (ThermoFisher) and siRNA (final concentration of 20 nM) were prepared according to manufacturer’s instructions. siOCT4 or siB2M was transfected for 24 h, and medium was changed as shown in Fig. 2a. Cells were analyzed at 48 h or 72 h.

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