Project description:Six1, Six4 and Myogenin are transcription factors that are known to be required for skeletal myogenesis. Currently, very little is known about the genes targeted by Six1 and Six4. Gene expression profiling when one or both transcription factors were knock-down by siRNA was performed to identify genes affected by their absence. We also hypothesized that Six1 and Six4 can work in cooperation with the myogenic regulatory factor (MRFs) family of transcription factors, such as Myogenin. Therefore, we performed the same type of experiment where the myogenin was knocked-down by siRNA to identify genes that are possibly regulated by the Six1 or Six4 in conjunction with Myogenin. C2C12 Myoblasts were transfected with siRNA against Six1, Six4, Six1 with Six4, Myogenin, or control 24h before start of differentiation. The cells were allowed to differentiate in differentiation medium for 24h and were harvested for gene expression profiling. Four replicates per siRNA were performed.
Project description:Six1, Six4 and Myogenin are transcription factors that are known to be required for skeletal myogenesis. Currently, very little is known about the genes targeted by Six1 and Six4. Gene expression profiling when one or both transcription factors were knock-down by siRNA was performed to identify genes affected by their absence. We also hypothesized that Six1 and Six4 can work in cooperation with the myogenic regulatory factor (MRFs) family of transcription factors, such as Myogenin. Therefore, we performed the same type of experiment where the myogenin was knocked-down by siRNA to identify genes that are possibly regulated by the Six1 or Six4 in conjunction with Myogenin.
Project description:In this study, we used ChIP-seq to map Six4 binding profile in different C2C12 cell lines 24 hours after differentiation (T24). We performed ChIP-seq using two different antibodies: anti-Flag antibody in Flag-Six4 C2C12 cell line or in parental C2C12 cells; a custom-made anti-Six4 antibody in shNS C2C12 cell line (a control cell line) or shSix4 C2C12 (C2C12 with stable Six4 knockdown using short hairpin RNA). We also performed ChIP-seq in parental C2C12 cells using normal rabbit IgG. We were able to identify Six4-bound loci in C2C12 T24 that were recognized by two different antibodies and showed a decrease in peak intensity in shSix4 C2C12 compared to shNS C2C12 cells.
Project description:In this study, we used ChIP-seq to map Six4 binding profile in different C2C12 cell lines 24 hours after differentiation (T24). We performed ChIP-seq using two different antibodies: anti-Flag antibody in Flag-Six4 C2C12 cell line or in parental C2C12 cells; a custom-made anti-Six4 antibody in shNS C2C12 cell line (a control cell line) or shSix4 C2C12 (C2C12 with stable Six4 knockdown using short hairpin RNA). We also performed ChIP-seq in parental C2C12 cells using normal rabbit IgG. We were able to identify Six4-bound loci in C2C12 T24 that were recognized by two different antibodies and showed a decrease in peak intensity in shSix4 C2C12 compared to shNS C2C12 cells. We established a C2C12 cell line with stable Six4 knockdown by short hairpin RNA (shSix4) vs. a control cell line (shNS). We also established a C2C12 cell line with stable expression of Flag-Six4-myc by infection of retroviruses expressing pBABE-Flag-Six4-myc (Flag-Six4 C2C12) vs. parental C2C12. We differentiate these cells for 24 hours before using them for ChIP-seq.