GSM9064798: Hypothalamus_Paired-Tag_H3K27ac_H3K27me3_sub_library_DNA#01; Mus musculus; OTHER
[CUT&Tag ]  Hypothalamic samples were dissected and finely minced on ice. The tissue fragments were lysed in cold nuclear extraction buffer (20 mM HEPES–KOH, pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% glycerol, 0.5 mM spermidine (Sigma-Aldrich, 05292), and 1× cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail (Roche, 11836170001)) for 10 minutes. After centrifugation at 500 rpm for 10 minutes at 4 °C, the supernatant was carefully removed, and the nuclei were resuspended in PBS supplemented with 1% BSA. [CUT&Tag ] CUT&Tag was performed using a modified protocol. Nuclei were first incubated in cold antibody buffer (20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM spermidine, 1× cOmplete™ Mini EDTA-free protease inhibitor, 0.01%IGEPAL-CA630, 0.01% digitonin (Sigma-Aldrich, D141-100MG), 2 mM EDTA) containing anti-H3K27ac antibody (Abcam, ab7429) and pA-Tn5. The suspension was rotated for 1 hour and then incubated overnight at 4 °C. Following incubation, nuclei were washed twice with Digitonin300 buffer (20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM spermidine, 1× cOmplete™ Mini EDTA-free protease inhibitor, 0.01% digitonin), then incubated in tagmentation buffer (Digitonin300 buffer supplemented with 10 mM MgCl₂) for 1 hour at 37 °C. To terminate tagmentation and release DNA fragments, nuclei were lysed in lysis buffer (10 mM EDTA, 0.1% SDS, 20 mg/mL Proteinase K) and incubated for 1 hour at 50 °C. DNA was purified by phenol-chloroform extraction. Libraries were amplified with 6 PCR cycles and purified using 0.8× AMPure XP beads (Beckman Coulter, A63881). Sequencing was performed on an Illumina NovaSeq 6000 platform using 150 bp paired-end reads. [Paired-Tag] Single-cell suspension were prepared from hypothalamus tissues in douncing buffer with Protease/RNase Inhibitor cocktail (0.25 M sucrose (Sigma, S7903), 25 mM KCl (Sigma, P9333), 5 mM MgCl2, 10 mM Tris-HCl pH 7.4, 1 mM DTT (Sigma, D9779), 1X Protease Inhibitor, 0.5 U/µL RNase OUT, 0.5 U/µL SUPERase Inhibitor) supplemented with 0.1% Triton-X100 (Sigma T9284). The cell suspension was then through a 30 µm Cell-Tric (Sysmex) and centrifuged at 1,000 g for 10 min, 4 °C. Cell pellets were washed with fresh douncing buffer, centrifuged again, and then resuspended in NIB containing 0.2% IGEPAL CA-630. The suspension was rotated for 10 minutes at 4 °C to lyse the cells and release nuclei. Nuclei were counted using a Bio-Rad TC20™ cell counter and immediately processed for Paired-Tag experiments. [Paired-Tag] Libraries were prepared using the Paired-tag workflow. Briefly, permeabilized nuclei were first incubated with antibodies targeting specific chromatin regions to enable the binding of protein A-fused Tn5 transposase. Tagmentation and reverse transcription were then performed sequentially. Specifically, reactions were carried out in 12 wells, each using the paired set of barcodes incorporated into the transposase adaptors and reverse transcription (RT) primers, thereby labeling different samples with the first round of barcodes. Next, a ligation-based combinatorial barcoding strategy was employed to introduce the second and third rounds of barcodes. This was achieved by sequentially ligating well-specific DNA barcodes—derived from 96-well plates—to the 5' ends of both DNA fragments and cDNA. Finally, the barcoded nuclei were divided into sub-libraries and lysed, after which DNA and cDNA were purified, amplified, and split into two separate sequencing libraries. [CUT&Tag ]  Hypothalamic samples were dissected and finely minced on ice. The tissue fragments were lysed in cold nuclear extraction buffer (20 mM HEPES–KOH, pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% glycerol, 0.5 mM spermidine (Sigma-Aldrich, 05292), and 1× cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail (Roche, 11836170001)) for 10 minutes. After centrifugation at 500 rpm for 10 minutes at 4 °C, the supernatant was carefully removed, and the nuclei were resuspended in PBS supplemented with 1% BSA. [CUT&Tag ] CUT&Tag was performed using a modified protocol. Nuclei were first incubated in cold antibody buffer (20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM spermidine, 1× cOmplete™ Mini EDTA-free protease inhibitor, 0.01%IGEPAL-CA630, 0.01% digitonin (Sigma-Aldrich, D141-100MG), 2 mM EDTA) containing anti-H3K27ac antibody (Abcam, ab7429) and pA-Tn5. The suspension was rotated for 1 hour and then incubated overnight at 4 °C. Following incubation, nuclei were washed twice with Digitonin300 buffer (20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM spermidine, 1× cOmplete™ Mini EDTA-free protease inhibitor, 0.01% digitonin), then incubated in tagmentation buffer (Digitonin300 buffer supplemented with 10 mM MgCl₂) for 1 hour at 37 °C. To terminate tagmentation and release DNA fragments, nuclei were lysed in lysis buffer (10 mM EDTA, 0.1% SDS, 20 mg/mL Proteinase K) and incubated for 1 hour at 50 °C. DNA was purified by phenol-chloroform extraction. Libraries were amplified with 6 PCR cycles and purified using 0.8× AMPure XP beads (Beckman Coulter, A63881). Sequencing was performed on an Illumina NovaSeq 6000 platform using 150 bp paired-end reads.