Identifying differential binding of transcription factor CLAMP on chromatin in S2 (male) and Kc (female) emmbryonic cell lines

CLAMP is a maternally deposited transcription factor binding to GA-rich genomic region. It recruits male-specific MSL (Male sex-lethal) complex on male X-chromosome that regulates dosage compensation in males, and competes with other GA-binding proteins like GAF, acting both synergistically as well as antagonistically in a context dependent manner. Considering CLAMP's male specific role in dosage compensation but ubiquitous expression in both males and females, what regulates differential binding of CLAMP is still unknown. Furthermore, ChiP-seq analysis has being long used to study protein-DNA interactions, however, since it uses physical force to shear DNA after protein capture, it is prone to both false negative and false positive peaks being called. To minimise these discrepancies, a recent CutnRun MNase based technique have being developed (Heinkoff Lab) which captures the DNA fragments bound to protein of interest. Due to higher accuracy in identifying the size and sequence of the protein bound DNA region, one can also identify monomeric or multimeric protein binding on the chromatin. We have used this technique to identify differential and shared CLAMP binding sites in male and female cells. We also compared CLAMP DNA binding peaks with CLAMP RNA binding peaks (iCLIP data) in male and female cells to understand sex-biased co-transcriptional RNA processing mechanism. rabbit anti-CLAMP was used to immunoprecipitate CLAMP bound DNA fragments from non-UV crosslinked, unfixed male (S2) and female (Kc) cell lines. 3 replicates each for males and females were run, however, during later stages one female sample was dropped due to insufficient starting material. Rabbit IGG was used as control, one for each male and female cell line sample.