Illumina HiSeq 2500 sequencing; Germinal Center B-Cell gene expression comparison between Wiskott–Aldrich syndrome protein KO mice and WT mice
Germinal Center B-Cell gene expression comparison between Wiskott–Aldrich syndrome protein KO mice and WT mice
Spleens were recovered immediately after euthanasia, perfused with complete cold medium and cut in small pieces to be crushed on a 70µm filter. Cold ACK was used for red blood cell lysis and cells were kept on ice in medium or complete medium. For surface staining, cells were incubated 30mins on ice with antibodies. Dark zone germinal center B cells sorted as DAPI-CD19+ GL7+IgD-CXCR4highCD86low RNEasy (qiagen) Kit manufacturer protocol. RNA quality was assessed on a Fragment Analyzer (Agilent Technologies). The RNAs had RQNs between 7.6 and 9.4. From 50 - 75 ng total RNA, mRNA was isolated with the NEBNext Poly(A) mRNA Magnetic Isolation Module. RNA-seq libraries were then prepared from the mRNA using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). Libraries were quantified by a fluorimetric method and their quality assessed on a Fragment Analyzer (Agilent Technologies).