B Cell RNA Isolation and cDNA Preparation

B-cells only or B-cells together with cT_FH cells were cultured for 6–7 days as described above. Transcriptional analysis of the co-cultured B cells used samples from 3 naïve animals, collected 4 weeks prior to the first prime vaccination. B-cells were collected from individual wells, positively selected using PE-CD19 (Beckman Coulter) and anti-PE magnetic beads (Miltenyi) and lysed by adding RLT buffer. RNA was extracted using the AllPrep DNA/RNA Micro Kit (Qiagen, Valencia, CA, USA). Each homogenized cell lysate was transferred to an AllPrep DNA spin column, and the flow-through was collected for RNA purification. An equal volume of 70% ethanol was added to the flow-through, mixed by pipetting, and transferred to an RNase MinElute spin column. The column flow-through was discarded and the column was washed with RW1 buffer. The spin column was further washed by RPE buffer followed by 80% ethanol. Finally, RNase free water was added to elute the RNA. RNA was measured by NanoDrop and 25 ng RNA was used for cDNA preparation with the RT2 First Strand kit (Qiagen, Valencia, CA, USA). The RNA was treated with genomic DNA elimination mix for 5 min at 42°C followed by 1 min incubation on ice. Equal amounts of reverse-transcription mix and the RNA/genomic DNA elimination mix were gently mixed by pipetting and incubated at 42°C for 15 min. The reaction was stopped by incubating at 95°C for 5 min. The first strand cDNA was diluted with RNase free water and used for Real-Time PCR.