GSM7814261: WT B cells (IgM+, IgD-) Biological Replicate 1; Mus musculus; RNA-Seq
Single-cell suspensions of thymi from 5 mice per genotype were incubated with LIVE/DEAD fixable dye (Invitrogen, 1:500) and mouse Fc Block (1:100) in PBS for 15 min on ice. Cells were washed with fluorescence-activated cell sorting (FACS) buffer (2% FCS in PBS) and incubated with fluorochrome-conjugated antibodies (1:300) for 25 min on ice in the dark. For each sample, 2000 cells were sorted into 10 µl TCL Buffer (Qiagen). For WT samples, the sort-gate was set to viable CD3-CD4-CD8a-NK1.1-CD11b-F4/80-CD19+B220+IgM+IgD- cells, representing the thymic B cell population known from other experiments to have the highest Aqp4 expression. For CD40KO samples, the thymic B cell population diversity was strongly diminished, containing virtually no IgM+IgD- cells, requiring us to sort the entire thymic B cell population for comparison, i.e. with the sort gate set to viable CD3-CD4-CD8a-NK1.1-CD11b-F4/80-CD19+B220+ cells. RNA was isolated from TCL lysate via AmpureXP beads (Beckman Coulter). Library preparation for bulk-sequencing of poly(A)-RNA was done as described previously (Parekh et al., 2016, Sci Rep). Briefly, barcoded cDNA of each sample was generated with a Maxima RT polymerase (Thermo Fisher, #EP0742) using oligo-dT primer containing barcodes, unique molecular identifiers (UMIs) and an adaptor. 5'-ends of the cDNAs were extended by a template switch oligo (TSO) and full-length cDNA was amplified with primers binding to the TSO-site and the adaptor. NEB UltraII FS kit was used to fragment cDNA. After end repair and A-tailing a TruSeq adapter was ligated and 3'-end-fragments were finally amplified using primers with Illumina P5 and P7 overhangs. In comparison to previous descriptions (Parekh et al., 2016, Sci Rep), the P5 and P7 sites were exchanged to allow sequencing of the cDNA in read1 and barcodes and UMIs in read2 to achieve a better cluster recognition. The library was sequenced on a NextSeq 500 (Illumina) with 65 cycles for the cDNA in read1 and 19 cycles for the barcodes and UMIs in read2.