GSM9131905: Adrenalitis (GEX library); Mus musculus; RNA-Seq
At day 14 after the immunization, adrenals were isolated, cleaned of surrounded fat and digested in 1.5 ml microtubes containing Collagenase D (0.5 mg/ml) and DNase I (0.1 mg/ml) (both from Sigma-Aldrich) in RPMI medium. Glands were incubated at 37 °C for 5 minutes, then minced with scissors, followed by an additional 20-minute incubation with continuous shaking. During incubation, tissues were gently disrupted at least once by reverse pipetting using wide-bore 1.5 ml tips. Following digestion, samples were transferred to 24-well tissue culture plates, and residual tissue fragments were passed through a 70 µm nylon mesh using syringe plungers. Cells were washed with 1× HBSS supplemented with 2% FBS and 200 mM EDTA and stained with fluorochrome-conjugated primary antibodies (CD11b-BV421, TCRb-FITC, CD4-PE). Each sample was labelled with Totalseq-C Hashtag antibodies for multiplexing (Biolegend, MH1: #155861, MH2: #155863, MH3: #155865, MH4: #155867, MH5: #155869, MH6: #155871, MH7: #155873, MH8: #155875, MH9: #155879, MH10: #155879). CD4+ T cells were sorted as live, CD11b-, TCRb+, CD4+ cells using MoFlo Influx sorter. The viability of the cells pre-loading was validated using TC20 Automated Cell Counter (#1450102, Bio-Rad) and was over 90%. Cells were loaded onto a 10X Chromium machine (10X Genomics). The libraries were constructed using the Chromium Single Cell 5' Library & Gel Bead and Chromium Single Cell 5' Feature Barcode Library kits (10X Genomics, #PN-1000014, #PN-1000020, #PN-1000080, #PN-1000009, #PN-1000084, #PN-1000071) following the Feature Barcode technology for Cell Surface Protein protocol (#CG000186 Rev D). The generated cDNA libraries were sequenced on NovaSeq 6000 (Illumina) using the S1 Reagent Kit (100 cycles, Illumina, #20012865).