GSM8263030: Donor_6_snRNA-seq; Homo sapiens; RNA-Seq
The tissue was cut into as small blocks as possible by scissors, and digested with 2 mg/ml. collagenase IV (2091GR001, BioFRoXX, Guangzhou, China) in the presence of 0.1 mg/ml DNase (Roche, Basel, Switzerland) for 20 min and ground by gentleMACS Dissociatir (Miltenyi Biotec, Germany) to obtain a single-cell suspension. Afterward, the cells were filtered with a 70-um cell strainer (Corning). Mononuclear cells were isolated from Ficoll-Paque Plus (17-1440-03; GE Healthcare) density gradient (lymphoprep; axis shield). Cells were lysed and nuclei were isolated according to the manufacturers' instructions: Nuclei Isolation for Single Cell Multiome ATAC + Gene Expression Sequencing protocol (CG000365 Rev A), and genomic DNA and RNA were processed with a Chromium Single Cell Multiome ATAC + Gene Expression kit refer to the following protocol: Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide (CG000338 Rev E). Single-cell RNA-seq and ATAC seq libraries were prepared in the Wuhan Biobank using the following reagents: Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent bundle (PN-1000283), Chromium Next GEM Chip J Single Cell Kit (PN-1000234). Nuclei were transposed in bulk solution in a Veriti 96-Well Thermal Cycler. The transposed nuclei suspensions were then loaded into a microfluidic chip and run in the Chromium Controller to generate Gel Beads-in-emulsion (GEMs) containing single nuclei. All steps to produce single-cell ATAC and gene expression libraries were performed according to the manufacturer's instructions. cDNA, ATAC library, and gene expression library quantity and quality were assessed using the Agilent 2100 Bioanalyzer (High Sensitivity DNA chip). Libraries were sequenced on a NovaSeq6000 instrument. Chromium Single Cell Multiome ATAC + Gene Expression