GSM7072950: serum_d12_ATACseq_rep1; Mus musculus; ATAC-seq
For RNA-seq, total RNAs were extracted with TRIzol® reagent. RNA sequencing libraries were constructed using the VAHTS mRNA-seq V3 Library Prep Kit (Vazyme Biotech). The libraries were denatured and diluted at a proper concentration, then were sequenced on Illumina NovaSeq (Annoroad Gene Technology Co., Ltd). For ATAC-seq, 50,000 cells were harvested and washed once with 50 μL cold PBS. Then the cells were resuspended in 50 μL lysis buffers (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.2% (v/v) IGEPAL CA-630). Then the suspension of nuclei was centrifuged 500 × g at 4°C for 10 min. The pellet was resuspended with 50 μL transposition reaction mix (10 μL TD buffer, 5 μL Tn5 transposase and 35 μL nuclease-free H2O), and incubated at 37°C for 30 min. Finally, DNA was isolated using MinElute PCR Purification Kit (QIAGEN). ATAC-seq libraries were constructed and were purified with AMPure XP beads (Beckman Coulter). The libraries were denatured and diluted, then were sequenced on HiSeq X-Ten (Annoroad Gene Technology Co., Ltd).  For ChIP-seq,1 × 107 cells were crosslinked with 1% formaldehyde at room temperature for 10 min. Then the reaction was stopped by adding glycine (final concentration, 0.125 M). Crosslinked cells were lysed in ChIP SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.0)) containing 1 × protease inhibitor cocktail and PMSF, then sonicated to achieve a chromatin sized of 200-400 bp. After sonication, the supernatant was diluted with IP buffer and then co-incubated with antibody–Dynabeads protein A/G (1:1 mixed) at 4°C overnight with rotation. Antibodies included：anti-YY1 antibody (Abcam, ab109237)，anti-SMC1 antibody (Bethyl Laboratories, A300-055A).ChIP For Biotin ChIP, cells stably expressed biotin-alone or biotin-TEAD2 were expanded and crosslinked with 1% formaldehyde. Crosslinked cells were sonicated and diluted tenfold with ChIP dilution buffer, and then incubated with M280 streptavidin dynabeads at 4°C for overnight. Streptavidin dynabeads-bound DNA was subsequently washed twice with wash buffer 1 (2% SDS), once with wash buffer 2 (50 mM HEPES (pH 7.5), 1 mM EDTA, 500 mM NaCl, 0.1% sodium deoxycholate, 1% Triton X-100), once with wash buffer 3 (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate) and then twice with TE wash buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). ChIPed DNA was reverse-crosslinked and purified for DNA library construction followed by sequencing. For BL-HiC, the cells were treated with 1% formaldehyde at room temperature for 10 min followed by quenching with 0.2 M glycine. Nuclei were extracted for subsequent experiments, including digestion with HaeIII (NEB), end-plus-A treatment, proximity ligation with biotin-labeled Bridge Linker, DNA purification and enrichment of biotin-labeled DNA with Dynabeads M-280 streptavidin. The enriched bead-bound DNA was subjected to end repair, adapter ligation, PCR amplification and DNA library construction, followed by sequencing on the Illumina NovaSeq platform (Annoroad Gene Technology Co., Ltd.). For QHR-4C, digested suspensions of 1 × 105 - 1 × 106 cells from tissues were cross-linked with 2% formaldehyde for 10 min and terminated by 0.2 M glycine. The cell pellet was permeabilized and digested with DpnII overnight followed by proximity ligation. Then, DNA under 1000 bp was extracted and sonicated. To enrich ligation events associated with a specific viewpoint, an appropriate amount of sonicated DNA was taken as a template to linearly amplify for 100 cycles using a 5' biotin-labeled probe of the viewpoint of interest. The amplification products were incubated at 95°C for 5 min, immediately cooled on ice to obtain amplified ssDNA and then enriched with Dynabeads M-280 streptavidin. The bead-bound ssDNA was then ligated with adapters. Finally, QHR-4C libraries were constructed with specific primer pairs (forward primers containing Illumina P5 with sequences near a specific viewpoint and reverse primers containing Illumina P7 with an index and sequences matching the adapter) and then sequenced on the Illumina NovaSeq platform (Annoroad Gene Technology Co., Ltd.). RNA sequencing libraries were constructed using the VAHTS mRNA-seq V3 Library Prep Kit for Illumina (Vazyme). ATAC libraries were constructed using the VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme). BL-Hi-C libraries were constructed into standard illumina libraries. ChIP-seq libraries were constructed using the VAHTSTM Universal DNA Library Prep Kit for Illumina® V2. QHR-4C libraries were constructed into standard illumina libraries.