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The CUT&Tag assay was conducted with several adaptations using the Hyperactive Universal CUT&Tag Assay Kit for Illumina (TD903, Vazyme Biotech). Initially, 5x10^5 primary T cells were isolated and coupled to ConA beads for 10 minutes. Subsequently, these bead-bound cells were incubated overnight at 4 deg C in 50 ul of antibody buffer containing 2 ug of anti-STAT5 (D2O6Y) antibody. Afterward, anti-mouse IgG was added and incubated for an additional hour. The cells were then washed and incubated with 0.04 uM biotinylated-pA/G-Tnp for 1 hour, followed by suspension in tagmentation buffer TTBL for another hour. The reaction was halted by the addition of Proteinase K and 10% SDS at 55 deg C for 15 minutes. A spike-in mix (1pg/1x10^5 cells) was incorporated for subsequent data normalization. Post the stopping step, tagmented DNA was captured and isolated using streptavidin magnetic beads. Library construction was performed directly on the beads using the NEBNext Ultra DNA Library Prep Kit for Illumina (E7370S, NEB). The completed CUT&Tag libraries were sequenced on the Illumina NovaSeq 6000 platform in PE150 mode by Novogene, Beijing, China. |
ChIP-Seq |
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PAIRED
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