GSM8899167: IgG CUT&Tag in iGMPs Replicate 1; Mus musculus; OTHER
CUT&Tag for H3K27me3 in iGMPs was performed as previously described, using EpiCypher's CUTANA CUT&Tag Kit (14-1102; Kaya-Okur et al. 2019). Briefly, 200,00 cells were incubated in Nuclei Extraction Buffer (21-1021, 1x protease inhibitor, 0.5 mM spermidine) for 10 min on ice. Nuclei were spun and resuspended in Nuclei Extraction Buffer and then bound to concavalin A beads (21-1401, EpiCypher) and incubated for 10 min at room temperature. After binding, supernatant was removed, and nuclei were resuspended in Antibody Buffer with primary antibody and then incubated overnight at 4 °C. The following antibodies were used: H3K27me3 (1:50, 13-0055, EpiCypher) and IgG (1:50, 13-0042, EpiCypher). After primary antibody incubation, nuclei were washed (Wash Buffer 1) and then incubated with 1:100 Anti-Rabbit Secondary Antibody (13-0047) for 30 min at room temperature. Nuclei were then washed twice with Wash Buffer 1 and resuspended in Wash Buffer 2 for incubation with 1:20 pAG-Tn5 (15-1017) for 1 h at room temperature. Nuclei were then washed twice with Wash Buffer 2 and resuspended in Tagmentation Buffer (Wash Buffer 2, 10 mM MgCl2) and tagmented for 1 h at 37 °C. Nuclei were then washed with Pre-Wash Buffer and then SDS Release Buffer was added to each sample and incubated for 1 h at 58 °C. Reactions were quenched with SDS Quench Buffer. Samples were indexed with i5 and i7 Primers performing PCR according to manafacturer's recommendations and DNA was purified with SPRIselect beads (21-1405). Indexed samples were sequenced PE150 on a NovaSeq X Plus at the University of California, Irvine Genomics Research and Technology Hub.
GEO
GSM8899167_r1