Transcriptional profiling of wildtype versus Themis2-deficient follicular B cells. Transcriptional profiling of wildtype versus Themis2-deficient follicular B cells

This RNA-seqexperiment was designed to find transciptional differences between wildtype and Themis2-deficient B cells either directly ex-vivo or stimulated for 6 h with various stimuli in vitro. It is part of a larger study on the function of Themis2 in B cells. Therefore splenic, live, B220+ CD93- IgM+ CD23+ follicular B cells were sorted by flow cytometry from wildtype or Themis2-deficient (Themis2KO/KO) C57BL/6J mice. Unstimulated samples were were lysed directly after the sort. Stimulated samples were stimulated in vitro for 6 h at 37 degree Celsius at a concentration of 3 million cells/mL with either 10 microgram/mL anti-IgM or 10 microgram/mL LPS or 1 microgram/mL CD40L with 0.1 microgram/mL IL-4 and then lysed. RNA was extracted using Trizol reagent (Life Technologies) and cleaned up using the RNEasy Mini Kit (Quiagen). Single end, unstranded, poly-A-enriched libraries were made using the TruSeq RNA sample preparation kit (Illumina). Samples were analysed with an Illumina HiSeq 2000, collecting 13.2 _ 76.1 million reads of 75 bases per sample.