title: GSM8471212: mouse_lung; Mus musculus; OTHER
library_name: GSM8471212
library_construction_protocol: Fresh tissue was isolated from an 8-week-old ICR (CD-1®) mouse following IACUC-approved protocols. The harvested tissues included one cerebral cortex hemisphere (65 mg), one half of a kidney (45 mg), and one lung lobe (50 mg), which were immediately snap-frozen on dry ice. The frozen tissues, along with 75 µL of RNase Inhibitor V2 (S2 Genomics, 100-288-916), were then placed in the Dissociation Chamber of a pre-cooled standard Nuclei Isolation Cartridge (S2 Genomics, 100-063-287) and inserted into the pre-cooled Singulator 100TM (S2 Genomics, 100-060-817). Nuclei were isolated from the tissues using the Low Volume Nuclei Isolation protocol. Following isolation, the nuclei suspension was recovered from the Singulator cartridge and centrifuged at 500 rcf for 5 minutes at 4°C. The supernatant was removed, and the kidney and lung nuclei pellets were resuspended in 1 mL of Nuclei Storage Reagent (S2 Genomics, 100-063-405) supplemented with RNase inhibitor (1 U/µL). The brain nuclei were further processed using Debris Removal Stock Reagent (S2 Genomics 100-253-628) to eliminate myelin debris, according to the manufacturer's instructions. Finally, the nuclei were counted, and their viability was determined using AO/PI staining with the Nexcelom K2. The nuclei samples were subsequently fixed via the TempO-LINC Fixation protocol as described below, and stored at -80oC. Nuclei were fixed using the TempO-LINC Fixation Kit (BioSpyder Technologies Inc., cat# 202392). Fixed, cryopreserved mouse nuclei were thawed and barcoded with the TempO-LINC barcoding protocol. Briefly, nuclei were hybridized with the Mouse Whole Transcriptome v1.1 (30,162 probe pairs, BioSpyder, cat# 201186) DO pool and placed in an incubator overnight for 16 hours at 45oC.  Next, 100,000 nuclei were transferred and prepped for TempO-LINC Barcoding. Nuclei were equally distributed across the 48 wells of the TempO-LINC Barcode 1 plate at 2,083 nuclei/well. Following three rounds of ligation-based barcoding, nuclei were then distributed for direct PCR amplification at 1000 nuclei/well. PCR primers contained both Illumina sequencing adapters as well as dual indices that also served as the fourth TempO-LINC barcode.
identifiers.namespace: GEO
identifiers.id: GSM8471212_r1