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ChIP-seq of NFATC1's role in AhBMSCs

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE145961 | GEO | 2022/10/10

REPOSITORIES: GEO

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Project description:Purpose: The ultimate goal of this study is the identification of transcription elongation-related chromatin remodeler and its in vivo mechanism in fission yeast through Next-generation sequencing (NGS) based ChIP-seq technique. Methods: Every ChIP-seq cells were grown up to mid-log phase and harvested after final 1% formaldehyde fixation and 125mM glycine quencing. Cells were harvested by centrifugation at 4 Celcius degrees and the cell pellets were washed by 15mL of pH7.5 TBS buffer per a conical tube. ChIP cells were disrupted by glass bead vortexing in ChIP lysis buffer and sonicated by Sonic Dismembrator Model 500, Fisher Scientific. Debris of the sonicate was excluded by centrifugation by 14,800rpm at 4 Celcius degree. IP samples were aliqouted from the sonicate and immunoprecipiated by proper amount of antibody and protein A/G beads, washed by ChIP lysis buffer for over-night at 4 Celcius degrees. IP samples were washed and eluted by proper buffers in Sigmaprep spin column. Proteins in both IP and input samples were degraded by 2hr 30ug proteinase K rxn and de-crosslinked at 65 Celcius degrees for at least 8hrs. The IP and input DNA were eluted by Quiagen PCR purification kit. Results: RNAPII and factor ChIP-seq in various mutants of chromatin remodelers showed that FUN30 family promotes transcription at gene coding regions. RNAPII ChIP-seq in fft3Δ, spt16-1 and fft3Δspt16-1 demonstrated that RNAPII regulation function of Fft3 is largely overlapped with that of Spt16. Histone H3 ChIP-seq results in fft3Δ and spt16-18 demonstrated that Fft3 is major chromatin remodeler to promote nucleosome turnover through nucleosome disassembly. Finally, histone H3 and RNAPII ChIP-seq in fft3Δ, H3/H4DD and fft3Δ H3/H4DD were showed that Fft3 facilitates RNAPII elongation at ORF regions through nucleosome barrier regulation. Conclusions: Fun30-Fft3 is major chromatin remodeler to facilitate RNAPII elongation by nucleosome disassembly-driven nucleosome barrier alleviation in fission yeast.

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ChIP-seq of NFATC1's role in AhBMSCs

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