title: GSM7307034: hippocampus 1_Smad4_CST; Mus musculus; OTHER
library_construction_protocol: CUT&Tag assay was performed as described previously with modifications(Hatice et al., 2019 ). Briefly，  native nuclei were purified from frozen samples as previously described (Corces et al., 2017). Concanavalin A coated magnetic beads were added and incubated at room temperature for 10 min. Bead-bound cells were incubated with rabbit anti-Smad4 (1:50, Cell Signaling Technology, catalog 46535) or IgG control antibody (1:50, Millipore, catalog 12-370) on a rotating platform overnight at 4 °C. Secondary antibody (anti-Rabbit IgG antibody, Millipore, USA) was added and pA-Tn5 adapter complex was prepared in dig-med buffer. Then cells were resuspended in tagmentation buffer (10 mM MgCl2 in Dig-med Buffer) and incubated at 37 °C for 1 h. DNA was purified using phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation.   1. Hatice S. Kaya-Okur, Steven J. Wu, Christine A. Codomo, Erica S. Pledger, Terri D. Bryson, Jorja G.Henikoff, Kami Ahmad & Steven Henikoff. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nature communications. 2019, 10(1):1930.  2. Corces, M., Trevino, A., Hamilton, E. et al. An improved ATAC-seq protocol reduces background and   enables interrogation of frozen tissues. Nat Methods 14, 959–962 (2017) doi:10.1038/nmeth.4396 The size distribution of libraries was determined by Agilent 4200 TapeStation analysis, and libraries were mixed   to achieve equal representation as desired aiming for a final concentration as recommended by the manufacturer.   Sequencing was performed in the Illumina Novaseq 6000 using 150bp paired-end following the manufacturer's   instructions. Other: Cut&tag