GSM9085086: Estrogen receptor CUT&Tag, Estradiol treatment, replicate 2; Homo sapiens; OTHER
For the CUT&Tag procedure, cells were harvested, counted, and assessed for viability. The iDeal CUT&Tag kit (Diagenode C01070021) was employed with few modifications to the original protocol. CUT&Tag. In brief, 100,000 cells were permeabilized and immobilized on concanavalin A-coated magnetic beads. Cells were then incubated overnight at 4°C with primary antibodies targeting H3K27ac (1:50, Active Motif Cat. No. 39034), ER (1:50, Epicypher Cat. No. 13-2012) and IgG (Diagenode kit) as a negative control, followed by a 30-minute incubation with a rabbit secondary antibody. Next, loaded Tn5 was added, and the cleaved DNA was purified using the silica membrane column method. Library preparation was performed with Nextera™-Compatible Multiplex Primers (Active Motif, 53155), employing 14 PCR cycles. Post-PCR cleanup was carried out using AMPure XP Beads (Beckman Coulter, A63881). Library concentration was measured with Qubit (Invitrogen), and fragments profile assessed using Tapestation (Agilent). Two biological replicates were generated for each experimental condition. Libraries were normalized, pooled, denatured and loaded with 2% PhiX v3 control spike-in (Illumina) for sequencing on Illumina NextSeq550 sequencer. Sequencing parameters were set to 2 x 38 bp.