GSM9466329: WT mouse iGC B cells replicates 3, Cut&Tag; Mus musculus; OTHER
Approximately 1×10^5 cultured iGCB cells were harvested, washed once with 500 μL Wash Buffer, and bound to Concanavalin A (ConA)-coated magnetic beads for 10 minutes at room temperature to immobilize cells. The assay was performed following manufacturer's instructions of the Hyperactive In-Situ ChIP Library Prep Kit for Illumina (pA/G-Tn5) kit (Vazyme, TD903). Bead-bound cells were permeabilized and incubated overnight at 4°C with primary antibody (Rabbit anti-Tcf3/TLE3, 11372-1-AP) and anti-rabbit IgG in Antibody Buffer containing 0.05% Digitonin. After washing, cells were incubated with a secondary antibody (Goat anti-rabbit IgG, Abcam ab6708) for 1 hour at room temperature. Following washes with Dig-wash Buffer, chromatin was tagged in situ by adding 0.05 μM Hyperactive pA/G-Tn5 Transposome and incubating for 1 hour at room temperature. Cells were washed with Dig 300 buffer, resuspended in tagmentation buffer, and incubated at 37°C for 1 hour. The reaction was stopped by adding Proteinase K, and DNA was purified using DNA clean beads. Purified DNA was amplified for 12-15 cycles using 2× CAM Mix and uniquely barcoded i5/i7 primers (TruePrep Index Kit V2, Vazyme TD202). Final PCR products were purified using VAHTS DNA Clean Beads (Vazyme N411) and eluted in nuclease-free water. Library quality was assessed by PCR and PAGE.