Lineage structure of the human antibody repertoire in response to influenza vaccination
10 million PBMCs or sorted cells with varying numbers lysed in RLT buffer was used as input material for RNA purification.  This was done by using the All prep DNA/RNA purification kit (Qiagen, Valencia, CA) following manufacture’s instruction. The concentration of the RNA was determined using a Nanodrop spectrophotometer.   cDNA was synthesized using SuperScriptTM III  reverse transcriptase (Invitrogen, Carlsbad, CA). One fifth of the RNA purified from each sample was used for cDNA synthesis reactions with a total volume of 60ul. All five constant region reverse transcription primers were added to the same reaction together with SUPERase•InTM (Ambion, Austin, TX). RNase H (Invitrogen Carlsbad, CA) was added to each reaction to remove RNA at the end of the cDNA synthesis step. All enzyme concentrations, reaction volumes and the incubation temperature were based on the manufacturer’s protocol for synthesis of cDNA using gene specific primers.   For each sample, 11 PCR reactions were set up corresponding to 11 forward primers with a mixture of 5 reverse primers in each reaction. 2ul of reverse transcription mixture was used in each PCR reaction of 50ul. Final concentration of 200nM was used for each primer. The PCR program began with an initial denaturation at 94°C for 2 minutes, followed by 35 cycles of denaturation at 94°C for 30 s, annealing of primer to DNA at 60°C for 30 s, and extension by Platinum® Taq DNA Polymerase High Fidelity (Invitrogen, Carlbad, CA) at 68°C for 2 minutes. PCR products were first cleaned using QIAquick PCR Purification Kit (Qiagen, Valencia, CA) and then purified using the QIAquick gel extraction kit (Qiagen, Valencia, CA). Concentration was measured using the nanodrop spectrophotometer.About 0.5µg of QIAquick cleaned PCR product for each sample was used to start the 454 library preparation process. 454 Titanium shotgun library construction protocol was followed for all samples. Briefly, double stranded DNA was end polished and ligated to sequencing adaptors which contained a molecular identifier (MID, a nucleotide based barcode system). The rest of the Roche 454 protocol was followed which includes library immobilization, fill-in reaction and single stranded template DNA (sstDNA) library isolation. The sstDNA was quantified using a digital-PCR method (28). Up to 16 libraries were pooled for one sequencing run and Roche 454 emulsion PCR and sequencing protocols were followed for the rest of the sequencing procedure.