GSM8280226: PAT, rep2, rep3, scRNA-seq; Mus musculus; RNA-Seq
Nuclei isolation from frozen mouse adipose tissue was performed following a modified protocol for single-cell multiome ATAC analysis. Briefly, frozen mouse adipose tissues were minced to prepare <0.5 cm pieces on dry ice. The pieces were transferred to a gentleMACS C tube (Miltenyi Biotec, Bergisch Gladbach, Germany) with chilled TST buffer, containing 0.03% Tween20, 0.01% BSA, 146 mM NaCl, 1 mM CaCl2, 21 mM Mg Cl2, and 10 mM Tris-HCl pH 7.5 prepared in ultrapure water. Tissues were then dissociated using the gentleMACS Dissociator (Miltenyi Biotec) by running the mr_adipose_01 program twice. The homogenates were incubated on ice for 10 min and filtered through a 40-µm cell strainer, with washing into a single-nucleus suspension. Nuclei were centrifuged at 500 ×g for 5 min at 4 °C. Each pellet was resuspended in an appropriate volume of chilled nuclei resuspension buffer, containing 0.04% BSA and 0.2 U/μL RNase inhibitor in PBS. Nuclei were further purified and enriched using anti-nucleus microbeads (Miltenyi Biotec). Viability and concentration of nuclei were determined using Countess 3 (Thermo Fisher Scientific) with trypan blue staining. The nuclei of each sample were incubated with a specific hash-tag antibody (TotalSeq™-A0451~8, BioLegend) for 20 min on ice and then washed thrice with staining buffer. The hash-tagged nuclei were resuspended in the appropriate buffer for downstream analysis and combined to multiplex three or four samples, each containing an equal number of nuclei. Single-cell RNA and ATAC-seq were performed using a Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Kit (cat# CG000338, 10x Genomics, Pleasanton, CA, USA) following the manufacturer's instructions. Briefly, following reverse transcription and cleanup, barcoded ATAC fragments, cDNA, and HTO were amplified with the pre-amplification mix with 1 µL of 0.2 µM HTO-targeting additive primer. Each sample was divided and used separately as input for ATAC and cDNA/HTO. ATAC and cDNA libraries were constructed following the manufacturer's protocol. For the HTO-derived cell hashing library, cDNA/HTO products, which were amplified with cDNA primers and HTO-targeting additive primer, were selected with SPRIselect reagent for subsequent library construction. ATAC, cDNA, and HTO libraries were processed using the MGIEasy Universal Library Conversion Kit (App-A) (cat# 1000004155, MGI, Shenzhen, China) following the manufacturer's instructions. The converted libraries were sequenced using a DNBSEQ-G400 (MGI).