ChIP-qPCR Analysis of ASCL1 Binding

Each ChIP experiment was performed in at least three independent biological samples and performed as previously described [24 (link)]. Briefly, 1 × 10⁶ GBM CSCs, over-expressing either ASCL1 or GFP as control, were cross-linked with 1% formaldehyde for 10 min at r/t and the reaction was quenched by glycine at a final concentration of 0.125 M. Cells were lysed in lysis buffer and chromatin was sonicated. Fifty μg of each sonicated chromatin sample were incubated o/n at 4 °C with the following antibodies: anti-IgG (Santa Cruz) and anti-MASH1 (Ascl1; BD Pharmingen). Immunoprecipitated DNA was analyzed by qPCR by using SYBR GreenER kit (Invitrogen). Values were normalized to those obtained with a non-immune serum and divided by input. The data shown represent triplicate qPCR measurements of the immunoprecipitated DNA. The data are expressed as (‰) express 1/1000 of the DNA inputs. All ChIP experimental values were normalized to those obtained with the relative input sample.