GSM9522409: HU5; Homo sapiens; RNA-Seq
Flash-frozen lung tissues were first chemically fixed to preserve cellular integrity and RNA content. The tissues were minced into small pieces using a sterile blade on a glass surface and transferred to the fixation buffer at a ratio of 1 mL per 25 mg of tissue. The samples were incubated at 4°C for 24 hours without agitation. Following fixation, tissues were washed with PBS and subsequently quenched with the quenching buffer provided in the kit to neutralize the fixative. For dissociation, the fixed tissues were enzymatically digested using a pre-warmed dissociation solution containing the Liberase enzyme. The reaction was performed using a gentleMACS Octo Dissociator (Miltenyi Biotec, Germany) to ensure single-cell suspensions were obtained. The resulting cell suspension was filtered through a 30 μm strainer to remove debris and washed with PBS before proceeding to downstream applications. The dissociated fixed cells were processed using the 10x Genomics Chromium Fixed RNA Profiling Reagent Kit (10x Genomics, Pleasanton, CA). The processed samples were then loaded onto the 10x Genomics Chromium X instrument, where individual cells were encapsulated into nanoliter-scale Gel Beads-in-emulsion (GEMs) along with reagents necessary for downstream processing. Inside each GEM, probes were ligated, and unique molecular identifiers (UMIs) and barcodes were incorporated into the transcripts. After breaking the emulsions, the barcoded probes were amplified via polymerase PCR, and sequencing-ready libraries were generated using the Chromium Fixed RNA Gene Expression Library Kit, followed by size selection and quality control assessments. The libraries were sequenced with ~200 million PE150 reads per sample on NovaSeq X Plus (Illumina, San Diego, CA). The sequencing reads were analyzed using Cell Ranger 7 (10x Genomics, Pleasanton, CA) and the human reference genome Chromium Human Transcriptome Probe Set v1.0.1 GRCh38-2020-A. The aggregation of the samples was performed using the cellranger aggr function, normalizing for the total number of confidently mapped reads across the libraries.