title: GSM8545067: Th2_Pik3cdE1020K_IgG_CUTnTag; Mus musculus; OTHER
library_construction_protocol: Tagmentation CUT&Tag/v3 Naïve (liveTCRb+CD4+CD8-CD44-CD62L+CD25-), in vitro Th1 polarized (liveCD4+) and Th2 polarized (liveCD4+) CD4 T cells from WT and Pik3cdE1020K/+ mice were sorted using an Aria Fusion Sorter (BD), as previously described and then subjected to CUT&Tag using the CUT&Tag/v3 protocol (dx.doi.org/10.17504/protocols.io.bcuhiwt6) [20] as indicated on isolated nuclei from naïve CD4 T cells and in vitro polarized Th1 and Th2 cells from WT and Pik3cdE1020K/+ mice. CUT&Tag utilized primary antibodies against CTCF (D31H2, Cell Signaling) and Rabbit mAB IgG XP Isotype Control (DA1E, Cell Signaling). All primary antibodies were diluted 1:50 with the corresponding guinea-pig anti-rabbit secondary diluted 1:100 accordingly. pA-Tn5 was sourced pre-loaded (C01070001, Diagenode). Primary antibody incubation occurred for 1 hr at RT with nutation and all other protocol steps as indicated in the native CUT&Tag protocol. Prepared libraries were normalized and pooled and prepared for sequencing via NovaSeq SP (Illumina) at the NHLBI Genomics Core.