title: GSM9203606: Wk18_Group2-5_GEM1_S6-2_GEX; Macaca mulatta; RNA-Seq
library_construction_protocol: N332-GT5, Apex-GT6 and 10E8-GT12 -specific B cells were isolated from peripheral blood samples collected from rhesus macaques immunized with N332-GT5, Apex-GT6 and 10E8-GT12 germline targeting immunogens. Groups 1, 2 and 3 were immunized with N332-GT5, 10E8-GT12 24mer or Apex-GT6 respectively. Groups 4 and 5 were immunized with N332-GT5 in combination with Apex-GT6 or 10E8-GT12 24mer, respectively. Finally, Group 6 received all three GT immunogens. Booster immunizations including the same set of immunogens delivered at the prime were given at wk10. Peripheral blood mononuclear cells (PBMCs) were collected at weeks 12 and 18 post immunisation. To identify antigen-specific B cells, samples were stained with tetramerized332-GT5, Apex-GT6 and 10E8-GT12 probes conjugated to fluorescent and TotalSeq-C oligo-tagged streptavidin. Surface staining included a panel of fluorescently labeled antibodies, along with one of ten different TotalSeq-C oligo-tagged antibodies per sample. These oligo-tagged antibodies targeted β2M and CD298 and served as sample-specific hashtags to enable multiplexing across animals. Cells were sorted on a FACSymphony S6 (BD Biosciences). N332-GT5, Apex-GT6 and 10E8-GT12 -specific memory B cells were collected into 1.5 mL tubes containing RPMI 1640 medium supplemented with 50% fetal bovine serum (FBS) and kept on ice for immediate downstream processing. Libraries were constructed according to the manufacturer's protocol using the Chromium Next GEM Single Cell 5' Reagent Kit v2.