title: GSM8865786: Con_3; Mus musculus; RNA-Seq
library_name: GSM8865786
library_construction_protocol: For RAN-seq:Total RNA was extracted using the RNeasy Plus Mini Kit. The first step involved synthesizing first-strand cDNA from  2 μg of total RNA using the iScript reverse transcription supermix. For snRNA-seq: Nuclei isolation was using low-retention microcentrifuge tubes (Fisher Scientific) to minimize nuclei loss throughout the procedure. Briefly, tissue samples were manually dissociated with fine spring scissors for 10 minutes, homogenized in TST solution, filtered through a 30-μm MACS SmartStrainer (Miltenyi Biotec), and pelleted by centrifugation at 500g for 10 minutes at 4ْC. The nuclei pellets were further then purified for single-nuclei enrichment and debris removal with Anti Nucleus MicroBeads (Miltenyi Biotec, #130-132-997) according to the manufacturer's guidelines. The nuclei were resuspended in 100 μL of resuspension buffer (1X PBS + 1% BSA). Dual-fluorescent staining was performed using ViaStainTM AOPI Staining Solution (Nexcelom Bioscience, #CS2-0106). The quality and quantity of the nuclei were assessed using a Cellometer-K2 Fluorescent Cell Counter (Nexcelom Bioscience) in both brightfield, and the PE/Texas Red® channel with a bandpass filter 610/10 (nuclei stained fluorescent red). Although slightly compromised, the nuclei yield was high. For RNA-seq: we used an Ion AmpliSeq Custom Panel containing 4,604 cancer- and immune-associated genes, designed by Thermo Fisher using Ion AmpliSeq Designer, as described previously 45. A total of 10 ng of RNA from each sample was used to prepare the cDNA library. The libraries were multiplexed, amplified using the Ion OneTouch 2 System, and sequenced on an Ion Torrent Proton System (Thermo Fisher). For snRNA-seq:For single-cell Gel Beads-in-Emulsion (GEM) generation, we utilized the Chromium X system (10X Genomics) droplet-based microfluidic technology, following the Chromium Next GEM Single Cell 5' Reagent Kits v2 (Dual Index) protocol. The standard 5′-10X Genomics workflow was applied to generate sequencing-ready libraries in all downstream steps.
identifiers.namespace: GEO
identifiers.id: GSM8865786_r1