CUT&Tag of fruit fly: Hr3
Librarty strategy: CUT&Tag.CUT&Tag experiments were conducted following the manufacturer's instructions using the Hyperactive Universal CUT&Tag Assay Kit for Illumina Pro (Vazyme, TD904). In brief, about 1 x 105 cells were collected from wing disc for each sample group (four biological replicates). Cell nuclei were extracted using 100 l NE buffer per sample on ice for 10 min. After centrifugation, Extracts were resuspended in 100 l Wash Buffer and incubated with Concanavalin A beads Pro at room temperature for 10 min. The beads were incubated with primary antibody anti-HA tag (CST, 1:100) diluted in 50 l Antibody Buffer overnight at 4. Then, samples were incubated in secondary antibody diluted in Dig-Wash Buffer (1:100) and incubated at room temperature for 1 h. For each sample, Hyperactive pA/G-Transposon Pro (0.4 M) diluted in Dig-300 Buffer were added and incubated at room temperature for 1 h. After that, samples were washed with Dig-300 Buffer. For fragmentation, Samples were incubated with 40 l Dig-300 Buffer, 10 l TTBL at 37 for 1 h. About 1 pg spike-in was added for internal control before DNA extraction. Then, DNA was extracted by incubating with DNA Extract Beads Pro diluted in 50 l 3 x B&W Buffer at room temperature for 20 min. The i7 and i5 Indexed Primer were used for PCR amplification with the recommended cycle number according to the instructions. Finally, the CUT&Tag libraries passing fragment analyzer (Fragment Analyzer-12 / 96) quality control were then sequenced on Illumina NovaSeq 6000.
SUB15966538
exp3