GSM8735059: Control-7, snRNA; Homo sapiens; RNA-Seq
Tissue sections were frozen and stored at -80°C. 20 mg frozen tissues were thawed in 1 mL cold homogenization buffer (260 mM sucrose, 30 mM KCl, 10 mM NaCl2, 20 mM Tricine-KOH pH 7.8, 1 mM DTT, 0.5 mM Spermidine, 0.2 mM Spermine, 0.3% NP40, cOmplete Protease inhibitor (Roche), and Ribolock) and homogenized in a pre-chilled Dounce. Cell lysates were passed through a 70 µm Flowmi cell strainer before separation using a discontinuous iodixanol gradient and centrifuge at 1480 g at 4°C for 20 min in a swinging bucket centrifuge with the brake off. The nuclei band located at the interface between 30% and 40% iodixanol was collected and washed in RSB-T wash buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20) Libraries were generated using the 10x Genomics Chromium Single Cell Multiome ATAC + Gene Expression kit following the manufacturer's instructions, with the following modifications. Per sample, 16,100 nuclei were resuspended in 1x diluted nuclei buffer (10x Genomics) with 2% BSA (Sigma) with a capture target of 10,000 nuclei. ATAC libraries were sequenced to target of 25,000 read-pairs per nucleus and RNA libraries were sequenced to 20,000 read-pairs per nucleus on an Illumina NovaSeq 6000 instrument at the Florida State University Translational Science Laboratory. The matched RNA and ATAC libraries were processed using the 10x Genomics Cell Ranger ARC (cellranger-arc-2.0.0) pipeline with default parameters and aligned to the hg38 human genome assembly (refdata-cellranger-arc-GRCh38-2020-A-2.0.0).