title: GSM8977404: aged_d14_1; Mus musculus; RNA-Seq
library_construction_protocol: Tibialis anterior muscles were harvested, minced, and nuclei were isolated using two approaches: (1) Dounce homogenization in Miltenyi Nuclei Extraction Buffer with RNase inhibitor, followed by filtration and centrifugation; or (2) the 10X Genomics Chromium Nuclei Isolation Kit, following the manufacturer's protocol for frozen tissues. In both methods, nuclei were resuspended in wash buffer containing BSA and RNase inhibitor. Nuclei quality and counts were assessed by trypan blue exclusion with a hemocytometer or automated fluorescence-based cell counting. All steps were performed on ice or at 4°C to preserve RNA integrity. Single-nucleus RNA-seq libraries were generated using the 10x Genomics Chromium Single Cell 3' Gene Expression v3.1 chemistry. Nuclei were loaded onto a Chromium Controller to create gel bead-in-emulsions (GEMs) for barcoding and reverse transcription. Libraries were constructed following the manufacturer's protocol, either using the Standard or High-Throughput workflows. Some samples were multiplexed using 10x Genomics CellPlex Cell Multiplexing Oligos (CMOs) according to the CellPlex Protocol #1. Final libraries were sequenced on an Illumina NovaSeq X platform using a 2 × 150 bp configuration, targeting a depth of 50,000 reads per nucleus.
identifiers.namespace: GEO
identifiers.id: GSM8977404_r1