GSM8185638: hCNCC_TCF7L2_rep2; Homo sapiens; OTHER
The CUT&RUN assay was conducted in hCNCCs using the ChIC/CUT&RUN Kit (EpiCypher, catalog no. 14-1048). Approximately 500,000 cells were harvested using Accutase and subsequently incubated with activated ConA Beads for 10 minutes at room temperature. Primary antibodies were then added to each 50 µL reaction: IgG (EpiCypher, 13-0042k, 0.5 µg), H3K27ac (Active Motif, 39133, 0.5 µg), TCF7L2 (Cell Signaling, 2569S, 1 µg), and the mixture was incubated overnight on a nutator at 4°C. On the second day, pAG-MNase and cold calcium chloride were used to activate the cleavage of target DNA. E. coli Spike-in DNA (0.1 ng) was also added for normalization in downstream analyses. DNA purification involved phenol/chloroform extraction and ethanol precipitation. Library preparation for histone proteins was performed using the CUT&RUN Library Prep Kit (EpiCypher, 14-1001). For TFs, modified procedures, as previously reported (https:// doi.org/10.17504/protocols.io.bagaibse), were employed to enhance the preservation of small DNA fragments