title: GSM8801447: Mouse single-cell RNA sequencing Samples 1; Mus musculus; RNA-Seq
library_construction_protocol: Mouse peripheral blood underwent erythrocyte lysis and leukocytes were centrifuged and collected immediately. Cells were stained with 2 μL DAPI  (564907, BD Pharmingen) and 2 μL Draq5 (62251, Thermo Scientific) for 5 min. Cells were diluted to 4 mL for sorting (FACSMelody, BD Biosciences), and viability was verified by ethidium  homodimer (EtHD) staining (E1169, Thermo Fisher Scientific). Cells were processed for scRNAseq using the 10X fixed sample preparation kit (1000414, 10X Genomics). Cells were  pelleted and resuspended in 1 mL fixation buffer and maintained at 4oC for 24 h. Cells were then collected at 850g at 5 min at RT and resuspended in 1 mL chilled quenching buffer. Cell concentration was determined by EtHD staining (Countess, Thermo Fisher Scientific) and then stored at -80 oC after addition of 100 μL enhancer and glycerol (10% final concentration). Cells  were processed by the Washington University McDonnell Genome Institute (MGI) for scRNAseq using the 10X genomics 16-plex format (10X Genomics CDG000527 Rev E). Cell barcoding on the 10X Chromium X, library construction, amplification and sequencing were performed, and scRNAseq FASTQ files obtained from the MGI were processed using 10X genomics cell ranger. pipeline for sequence quality, demultiplexing, and alignment. Processed FASTQ files were exported as H5 files. 10X Genomics platform was used, 16-plex kit, on a Chromium X, following standard droplet-based scRNA-seq library preparation