Chromatin immunoprecipitation DNA sequencing (ChIP-seq) for Zmiz1 in Neuro-2a (N2a) cells

Purpose: Zinc Finger MIZ-Type Containing 1 (Zmiz1) is a member of the PIAS family of protein and function as a transcriptional coactivator of Notch, Androgen Receptor (AR), p53, Estrogen Receptor (ER), and Smad3/4 . Here, we use Neuro-2a cell line to perform ChIP sequencing on Zmiz1 bound, direct or indirect to profile Zmiz1 associated genomic regulation. Methods: Neuro-2a (N2a) (ATCC, CCL-131) were cultured in DMEM-high glucose (Cytiva, SH30022) supplemented with 5 % FBS (Cytiva, SH3008803) and 50 U/ml Penicillin-Streptomycin (Thermo Fisher Scientific, 15070063) at 37 °C and 5% CO2. Zmiz1-HA was overexpressed in N2a cells and subsequently, ChIP DNA samples were obtained by using ChIP-IT Express Enzymatic Kit (Active Motif, 53009) and processed according to manufacturer specifications. Anti-HA-Tag Antibody (Cell Signaling, 3724S) was used for immunoprecipitation. Pull down DNA quantity was measured using Qubit DNA HS kit (Thermo Fisher Scientific, Q32851). The sequencing library was prepared using TruSeq ChIP Library Preparation Kit (Illumina, IP-202–1012). Ten nanograms of starting material were used for library preparation. Library quality and quantity were assessed using Agilent DNA 1000 chip (Agilent 5067-1504) and Qubit DNA HS kit respectively. Libraries were sequenced using the NextSeq1000/2000 P2 Reagents (200 Cycles) v3 (Illumina, 20046812) on a Nextseq1000/2000 system. Sequencing analysis was done using the ChIPSeq App from BaseSpace Labs (Illumina), which uses MACS2 for region enrichment and HOMER for motif analysis. Results: ChIP-seq peaks analysis identified thousands of peaks which are mostly located in intergenic regions followed by introns. Conclusions: We identify Zmiz1 bound DNA (direclty or indireclty) in Neuro-2a cells thus providing genomic regulation in neurodevelopmental events. Overall design: Zmiz1-HA plasmid was transfected in Neuro-2a cell followed by chromatin immunoprecipitation (ChIP enzymatic kit from Active Motif), library prepraration, and sequencing.