GSM6127490: SAN, Control, Yap; Mus musculus; OTHER
Nuclei from SAN tissues were incubated with concanavalin A (Con A)-coated magnetic beads (Bangs Laboratories, BP531) at room temperature for 10 min to immobilized nucleus on the Con A-coated magnetic beads. The bead-bound nuclei were then incubated with anti-H3K4me3 (Active Motif, 39159, 1:100) and anti-Yap1 (Novus, NB110-58358, 1:50) antibodies separately overnight at 4oC with rotation. Guinea pig anti-rabbit second antibody (Antibodies-Online, ABIN101961, 1:100) was diluted in 50 mL of wash buffer, and added into the nucleus to incubate for 30 min at room temperature. The pA-Tn5 adapter complex was diluted 1:200in 300-buffer and incubated with nucleus at room temperature for 1 h. Nucleus were then resuspended in tagmentation buffer at 37oC for 1 h for DNA fragmentation. After 1 h, 0.5M EDTA, 10%SDS, and 20mg/mL Proteinase K were added to terminate tagmentation and incubate for 10 min at 70oC. DNA was then purified using phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation and dissolve in 21 μL 1 mM Tris-HCl at pH 8 and 0.1 mM EDTA. The libraries were prepared by add 2 uL of 10uM barcoded i5 primer, 2 uL of 10uM uniquely barcoded i7 primers and 25 uL NEBNext HiFi 2X PCR Master mix (Biolabs, M0541L), and PCR was performed according to the conditions in the protocol.  AMPure XP beads (Beckman Coulter, A63880) were used for DNA purification. Libraries were sequenced on NextSeq 550 (Illumina, USA) at UTHealth Cancer Genomics Center, and 75-bp paired-end reads were generated. Fastp software was used to remove adaptor and low-quality reads. All reads produced by CUT&Tag of H3K4me3 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 transcription start site (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.