GSM6605095: adult mouse anti-CD3 sorted splenocyte short-read 10x scRNA-seq, replicate 3; Mus musculus; RNA-Seq
Spleens were removed by dissection, transferred to a sterile petri dish containing PBS with 5% FBS, and minced using surgical blades. Spleen fragments were then ground through 100 μm and 40 μm cell strainers, pelleted, and resuspended in the ACK lysis buffer as described in the thymus tissue collection section. Cell density and viability were determined using Countness II Cell Counter. Subsequently, splenocytes were resuspended at 1 million cells per ml of 0.04% BSA/PBS. In order to sort out the splenocyte T-cell subsets for constructing 10× GEM single-cell libraries, splenocytes were resuspended at 1 million cells per ml of FACS buffer (PBS with 1% FBS and 5mM EDTA).splenocytes were pre-incubated with 1ug mouse Fc blocker (BD 553141) per 1 million cells/100ul FACS buffer for 5 minutes, and incubated with an anti-mouse CD3e (clone 17A2, eFlour450, 2 ug/ml) and an anti-mouse CD11b (M1/70, Alexa 488, 2 ug/ml) on ice for 40 minutes.after incubation, cells were washed and resuspended in the FACS buffer containing Sytox Blue Live/Dead dye for cell sorting using FACS BD Aria. Viable, single cells were gated, and the CD11b-/CD3e+ cells were collected for the generation of 10× GEM single-T-cell libraries Single cells were captured in droplet emulsion using the Chromium Controller (10× Genomics, Pleasanton, CA), and GEM single-cell libraries were constructed according to the 10× Genomics protocol using the Chromium Single-Cell 30 Gel Bead and Library 3' V3 kit (10× Genomics, Pleasanton, CA). In brief, cell suspensions were diluted in PBS with 0.04% BSA to a final concentration of 1×106 cells/mL (1,000 cells per μL). Cells were loaded in each channel with a target output of 10,000 cells per sample. All reactions were performed in a C1000 Touch Thermal Cycler (Bio-Rad Laboratories, Hercules, CA) with a 96 Deep Well Reaction Module. Twelve cycles were used for cDNA amplification and sample index PCR. Amplified cDNA and final libraries were evaluated using a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) with a high sensitivity chip. paired-end ilimina next-seq