Whole transcriptome profiling of germinal center B-lymphocytes using a universal, integrated workflow of FACS cell sorting and TempO-Seq? assay

摘要

Next Generation Sequencing (NGS) workflows for transcriptionally profiling cells involved in complex tissue functions have been limited in identifying and isolating rare cells, preparing suitable samples, and sequencing assays. TempO-Seq? provides a robust, quantitative, and specific gene expression platform that does not require RNA extraction or reverse transcription. It targets short RNA-sequences, is not biased, or require polyadenylation, and is compatible with fixed cell samples. Assays are available for human, rat, mouse. We implemented an integrated cell suspension workflow that combines surface and intracellular (ic) staining with WT TempO-Seq assay (～27,000 mouse genes) followed by FACS-purification of selected cells and sequencing. We assayed suspensions of individual mouse Peyer Patch (PP) cells (6-8 PP, ～3x106 cells/mouse), profiling gene expression of GC B-cells. After sequential surface labeling, fixation and permeabilization, ic-staining, and TempO-Seq, GC B-cells (～2-3% of total cells) were FACS-purified based on B220 CD95 GL7 staining, then sequencing-ready libraries prepared of 100-cell aliquots/sample. The assay has an excellent replicate repeatability (R2=0.94 detecting 11,000 genes in cell lines). Sorted, GC B-cells appeared more heterogeneous, with repeatability R2=0.73-0.78 and 7,000-8,000 different genes measured. Data integrity was demonstrated by concordance of selected protein markers (B220, CD95) and RNA expression (B-cell lineage genes, Fas). We confirmed selective expression of genes associated with the GC including BCL6, CXCR4, immunoglobulin class-switched isotypes, and downregulation of the GL7 repressor CMP-Neu5Ac hydroxylase. Using ic-staining for the cell cycle progression biomarker Ki67, we found that only 40-45% of PP GC B-cells stained positive, presumably undergoing affinity maturation through somatic hypermutation. We measured differential profiles and these and other data will be presented.