GSM4969123: shWDR5_H3K4ac_CUT&Tag; Homo sapiens; OTHER
CUT&Tag(Cleavage Under Targets and Tagmentation) Kit for Illumina was purchased from Vazyme(TD901). The assay was performed according to the protocol. Briefly, 1 × 105 QBC-939 cells were washed twice with wash-buffer and incubated with Concanavalin A-coated magnetic beads (ConA beads) at room temperature for 15 min. Bead-bound cells were collected by magnet stand and resuspended in 50 μl of antibody-buffer. 1 μg of anti-H3K4ac antibody (39381, Active Motif) was added and incubated at room temperature for 2 hours with slow rotation. 1 μg secondary antibody(goat anti-rabbit IgG H&L, Ab206, Vazyme) was diluted in 50 μl of Dig-wash buffer and cells were incubated at room temperature for 50 min. The Hyperactive pG-Tn5 Transposon was diluted 1:100 in Dig-300 buffer and incubated with cells at room temperature for 1 h. Cells were then resuspended in Tagmentation buffer for DNA fragmentation. After 1 hour,  EDTA, SDS and Proteinase K were added to terminate tagmentation. DNA was then purified using phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation. Library amplification was also performed according to the protocol. 24 μl DNA was mixed with 1 μl TAE, 10 μl 5×TAB, 5 μl ddH2O as well as 5 μl i5 and i7 primers from TruePrep Index Kit V2 for Illumina (Vazyme, TD202). PCR program was set according to the protocol. VAHTS DNA Clean Beads (Vazyme) was used for DNA purification. Libraries were sequenced on Novaseq(Illumina, USA) at Novogene Science and Technology CO.,Ltd(Beijing,China) and 150-bp paired-end reads were generated. Fastp software was used to remove adapter and low-quality reads. All reads produced by CUT&Tag of H3K4ac were aligned to the hg19 human genome using BWA(Burrows Wheeler Aligner). MACS2 was used for peak calling. Genes with a peak within 3 kb of the TSS were considered target genes. An area of 6 kb surrounding each TSS was selected to get CUT&Tag profiles of H3K4ac using deepTools software. Annotation of peaks was performed using ChIPseeker.