ChIP-seq of NFATC1's role in AhBMSCs

We report the application of ChIP sequencing technology for high-throughput profiling of target-gene of NFATC1 in NFATC1 knockdown (NFATC1 RNAi) or widetype control (NC) adult human bone marrow mesenchymal stem cells (AhBMSCs).To knockdown NFATC1 in AhBMSCs we transfected cells with NFATC1 specific siRNAs and the scrambled (SCR) RNAs were ste as controls. AhBMSCs are derived from a 24-year-old healthy male human donor. 48h-post siNFATC1 or negative control siRNA transfection, AhBMSCs were subjected to ChIP sample preparation, respectively using Chromatin-Prep-Kit (MM_NF-17-10461, Millipore, CA, USA) for Chromatin dissection and HiSens-Chromatin-Immunoprecipitation-Kit (MM_NF-17-375, Millipore, CA, USA) for immunoprecipitation according to instruction manuals. Briefly, cells were fixed using 1% formaldehyde for 1min at room temperature to crosslink chromatin. Adding 125mM glycine to stop crosslinking. Then cells were washed w/ cold PBS twice and then lysed using lysis buffer supplemented with protease inhibitors cocktails, followed by centrifuged to obtain cell lysates in according to the manual. After cell lysis, purified nuclei were collected and then incubated with nuclease to get mononucleosomes. After sample preparation soluble chromatin was precleared by SCW washing buffer and subjected to immunoprecipitation as described in manufacture’s protocols. For immunoprecipitation, 1-2µg of antibody of anti-NFATC1 (Novus, Cat#NB300-620) was added. After immunoprecipitation, chromatin was washed, eluted, reverse-crosslinked and digested. Finally purified DNA was subjected to the high-throughput ChIP-sequencing (Novogene, China) after quality check. The data was analyzed by Novogene Inc (China). Every group consists of 3 replicates which were from 3 independent experimental repeats. Examination of target-genes which are trancript directly via NFATC1 in AhBMSCs.