The yielded amplicon products were purified using AMPure, and the resulting individual amplicon DNA libraries were clonally amplified by multiplex emulsion PCR followed by sequencing using the GS FLX pyrosequencing platform.
The amplicon mixture for each individual was purified using an Illustra GLX PCR DNA and Gel Band Purification Kit (GE Healthcare, Little Chalfont, UK, 2008 version).
Alternatively, erythrocyte-enriched fractions from 27 HIV-positive individuals were purified using a mix of magnetic beads coated with antibodies to CD4 and CD8 (BD Biosciences).
The individual proteins were purified using a similar double Ni-nitrilotriacetic acid procedure as previously described [ 66].
Individual reactions were purified using the BigDye XTerminator Purification Kit (Applied Biosystems) before being loaded on an Applied Biosystems DNA Analyzer 3730XL.
In brief, DNA from the tail tip of an individual mouse was purified using a commercial kit (Easy DNA kit, Invitrogen).
After cross-linking reversal and proteinase-K digestion, each individual IP was purified using QIA-quick PCR purification kit (Qiagen, Valencia, CA, USA) followed by elution with 50 μL of elution buffer.
Total cellular RNA was purified using Quick‐RNA MicroPrep (Zymo Research, Irvine, CA, USA) for each individual immune population sorted from each individual mouse.
Individual PCR products were purified using Millipore's Amicon Microcon-PCR Centrifugal filter devices (Millipore, USA), pooled in equimolar amount with Beckman Biomex FX system equipped with a DTX880 Multimode Detector (Beckman, USA), and sequenced using the GS FLX Titanium pyrosequencing kit (Roche Applied Science. USA).
Incubation was at 37 °C for 40 min. The RNA from individual reaction mixes were purified using the phenol-chloroform extraction method and analyzed by denaturing gel electrophoresis (7 M urea, 12% polyacrylamide).
