GSM9688021: 1669, microglia, MG IFNAR KO TBI, 7 days post injury; Mus musculus; RNA-Seq
Single-cell suspensions were prepared as described previously. Viable cells were counted and loaded at the recommended concentration into the 10x Genomics Chromium Controller using Chromium Single Cell 5′ chemistry reagents. Cells were partitioned into nanoliter-scale Gel Bead-in-Emulsions (GEMs) together with barcoded gel beads, reverse-transcription reagents, and partitioning oil. Within each GEM, cells were lysed and polyadenylated mRNA was captured by oligo(dT)-primed reverse transcription, generating barcoded full-length cDNA that incorporates a 10x cell barcode and Unique Molecular Identifier (UMI). GEMs were then broken, and the barcoded cDNA was purified and amplified by PCR (following the 10x Genomics Chromium Single Cell 5′ Reagent Kit user guide). Amplified full-length cDNA derived from polyadenylated transcripts was used for 5′ gene expression library construction with the Chromium Single Cell 5′ Library Construction Kit (10x Genomics). The cDNA was enzymatically fragmented and size-selected, followed by end-repair, A-tailing, adaptor ligation, and sample-index PCR to add P5/P7 Illumina sequences and dual indexes. Final libraries were quantified (e.g., by qPCR or fluorometry) and size-selected prior to sequencing. All steps were performed according to the manufacturer's protocol for Chromium Single Cell 5′ chemistry (v2, GEM-X, or the specific reagent kit version used in your experiment).