GSM7763719: Beijing-you control, replicate 1, scRNAseq; Gallus gallus; RNA-Seq
Fresh spleen tissues were mechanically minced in PBS with 5% FBS (HyClone, Logan, UT, USA), and a syringe plunger was used to grind the spleen fragments through 100-μm and 40-μm cell strainers (Corning, NY, USA). The filtrated liquid containing cells was centrifuged at 500 × g for 10 minutes, the supernatant was discarded, and the cells were washed twice with 5% FBS and then suspended in 1640/DMEM with 5% FBS. The cells were incubated on ice with mouse anti-chicken CD45-PE (0.1 mg/mL; SouthernBiotech, UAB, AL, USA) for 30 minutes and 7-AAD (5 μL/105 cells; BIOESTABLISH, Beijing, China) for 10 minutes. CD45+ cells were collected by flow cytometry. After obtaining the single-cell suspension, we detected the cell activity of all samples (the samples with cell activity >85%, single good dispersion, and low impurity content were collected). Subsequently, single-cell gel bead emulsions (GEMs) were prepared by microfluidic channels, and the cell concentration was controlled within 700-1200 cells/μL. In the GEM reaction system, cells were broken and lysed. The released mRNA and poly-dT primer sequence were activated by reverse transcriptase to generate full-length cDNA and amplify and construct the library. The StepOnePlus Real-Time PCR System was used for q-PCR to accurately quantify the effective concentration of the library (the index of the effective concentration of the library was not less than 10 nM). The qualified library was sequenced by the Illumina platform to obtain raw reads. According to the 10× transcriptome library structure, the data were intercepted to obtain Read1 and target Read2, containing barcode and UMI sequence information.