GSM8447696: Exp 1 Ln GC; Mus musculus; RNA-Seq
On day 18, lungs were removed and bronchial lymph nodes were dissected from the lung tissue under a stereo microscope. Single-cell suspensions from lung tissue were prepared using the Miltenyi gentleMACS Dissociator and digested with 0.5 mg/ml collagenase D and 20 mg/ml DNase I for 25 min at 37°C. Antigen-specific B cells were first enriched for CD45.1 expressing cells by magnetic sorting. For higher purity, cells were sorted on a Becton Dickinson ARIA II cell sorter as DAPI- CD4- CD8- CD19+ CD45.1+ CD45.2- B cells and either as CD138- CD38low GL7+ geminal center (GC) B cells or CD19low CD138+ plasmablasts directly into Trizol LS. RNA was isolated using the Qiagen RNeasy Micro Kit. Heavy chain immunoglobulin BCR libraries were prepared according to the protocol of Chudakov (Turchaninova et al., Nat Prot (2016) doi: 10.1038/nprot.2016.093).