RNA-seq analysis of freshly isolated germinal center B cells

Purpose: The goal of this study is to complare the transcitpional profiles of freshly isolated, splenic, murin GCBC and other activated and naive non-germinal center B cell populations. Methods: Cells were freshly isolated, bead purified and FACS Ari sorted . Sorted cells (1.5-3x106 cells per sample) were washed twice in cold PBS and RNA was prepared using shredder-columns (Qiagen; QIAshredder 79656) and the RNeasy Plus Mini kit (Qiagen) following the manufacturer's instructions. 3 independent RNA libraries per cell population were prepared using Illumina TruSeq Stranded mRNA sample preparation kit. The first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T oligo attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity is achieved by using dUTP in the Second Strand Marking Mix, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. The products are then purified and enriched with PCR to create the final cDNA library. The cDNA libraries are validated using KAPA Biosystems primer premix kit with Illumina-compatible DNA primers and Qubit 2.0 fluorometer. Quality is examined using Agilent Tapestation 2200.The cDNA libraries were pooled at a final concentration 1.8pM. Samples were sequenced using Illumina NextSeq 500 with 75bp paired-end reads and aligned to the mm10 genome using the STAR aligner. The number of uniquely aligned reads ranged from 31 to 77 million. Using an optimized data analysis workflow, Gene-level counts were determined using featureCounts and raw counts were analyzed for differential expression using the “voom” method in the “limma” R package. Results: After determining genes that were differentially expressed between splenic GCBC and activated B cells, we performed gene set enrichment analysis (GSEA). Differentially expressed genes were compared to several previously defined hypoxic gene signatures. Overall design: mRNA profiles of freshly isolated splenic murin germinal center B cells were generated using deep sequencing, in triplicate, using Illumina NextSeq 500.