Ntrna

A mixture of 4 different siRNA sequences specific to Kdm6a was used at a final concentration of 40 nM total RNA during transfection (1: AGUUAGCAGUGGAACGUUA, 2: GGACUUGCAGCACGAAUUA, 3: GGUACGGCCUACUGGAAUU, 4: CCACGUUGGUCAUACUAUA; ON-TARGETplus Mouse Kdm6a Set of 4 siRNA, Dharmacon, UK). A non-targeting siRNA sequence (ntRNA; Dharmacon) was used as control and co-transfection with pmaxGFP (Lonza, Switzerland) was performed in all cases for transfected neurons identification. The efficacy of siRNA targeting Kdm6a was assessed by electroporation of neurons using a 4D-Nucleofector X Unit and the corresponding P3 Primary Cell nucleofection kit (Lonza) according to the manufacturer’s instructions, followed by qPCR for Kdm6a after 16 h of knockdown. The same experimental design was used to analyze the effect of Kdm6a knockdown on Ngn3 gene expression. The transfection efficacy by electroporation was 15%, calculated as the percentage of GFP-expressing cells over total number. For morphometric analysis, neurons were transfected at 3 DIV with siRNA targeting Kdm6a or ntRNA using Effectene Transfection Reagent (Qiagen, Germany) according to the manufacturer’s instructions. After 16 h of knockdown, cultures were processed for axonal length measurement. The transfection efficacy by this method was 0.1%.

Neurons were transfected by electroporation or lipofection using a mixture of four different siRNA sequences targeting Kdm6a transcripts at a final concentration of 40 nM total RNA (ON-TARGETplus Mouse Kdm6a set of four siRNA, Dharmacon, UK); procedures and knockdown efficacy were previously described and demonstrated (Cabrera Zapata et al., 2021 (link)). A non-targeting siRNA sequence (ntRNA; Dharmacon) was used as a control, and co-transfection with pmaxGFP (Lonza, Switzerland) was performed in all cases for transfected neuron identification. For immunofluorescence, neurons were transfected by lipofection at 3 days in vitro (DIV) with target siRNA or ntRNA using Effectene Transfection Reagent (Qiagen, Germany) according to the manufacturer’s instructions, and after 18 h of knockdown, they were fixed and immunolabeled. For gene expression analysis and ChIP assays, neurons were transfected by electroporation before seeding with target siRNA or ntRNA using a 4D-Nucleofector X Unit and the corresponding P3 Primary Cell nucleofection kit (Lonza) according to the manufacturer’s instructions, seeded, and incubated for three DIV until processing for RNA isolation/ChIP reactions.

Neurons were transfected by electroporation or lipofection using a mixture of four different siRNA sequences targeting Kif21B, or a mixture of two different siRNA sequences targeting Ngn3 transcripts at a final concentration of 40 nM total RNA (Dharmacon, United States) and a non-targeting siRNA sequence (ntRNA; Dharmacon) was used as control. Co-transfection with pmaxGFP (Lonza, Switzerland) was performed for transfected neuron identification in all cases. According to the manufacturer’s instructions, for immunofluorescence, neurons were transfected by lipofection at 3 DIV with target siRNA or ntRNA using Effectene Transfection Reagent (Qiagen, Germany). For gene expression analysis, neurons were transfected by electroporation before seeding with target siRNA or ntRNA using a 4D-Nucleofector X Unit and the corresponding P3 Primary Cell nucleofection kit (Lonza) according to the manufacturer’s instructions, seeded and incubated for 3 DIV until processing for RNA isolation or western blot.