Illumina sequencing of Mouse Splenocytes from a recipient prime immunized with gp120-YU2.DG
Whole spleens were extracted from the mice, mechanistically crushed in PBS and subsequently filtered in a 70m Cell Strainer (Corning). The preparation underwent Red Blood Cell lysis (401301, Biolegend). cDNA was generated from Quick RNA Microprep (Zymo) extracted RNA of total splenic lymphocyte populations with the RevertAid reverse transcriptase (K1621, ThermoFisher) using Oligo-dT primers. For Initial PCR amplification, 3BNC117.W.T. VH fragments were amplified with the proofreading PrimeStarMAX polymerase (R045A, Takara) for 40 cycles. Amplicons were purified using AMPureXP beads (A63881, Beckman Coulter) at a 0.7:1 ratio. The subsequent 8 cycles PCR reaction was performed with the PrimeStarMax polymerase (R045A, Takara). Libraries were purified using AMPureXP beads at a 0.7:1 ratio. Combined libraries were loaded at 5pM with 25% PhiX control (FC-110-3001, Illumina). Sequencing was performed in a high-throughput MiSeq machine using either a v2 Nano reagent kit 2x250bp or a v3 reagent kit 2x300bp (MS-102-3003 and MS-103-1003, Illumina) at the Genomic Research Unit (GRU) at the Faculty of Life Sciences, Tel Aviv University.