Illumina NovaSeq 6000 paired end sequencing; Single-cell immune repertoire sequencing of bone marrow plasma cells following infection and immunizations
Single-cell immune repertoire sequencing of bone marrow plasma cells following infection and immunizations
LCMV Infection was performed i.v. (tail vein) with 2x10e6 FFU of clone 13 LCMV. OVA immunizations were administered every two weeks with 100ug OVA with 20ug Monophosphoryl lipid A  (MPLA) subcutaneous. hTNFR2 immunizations were performed with 5 administrations every two weeks with 10ug TNFR2 and 20ug MPLA.  Bone marrow plasma cells were isolated either using CD138+ FACS or MACS following infections or immunizations.  All experiments were performed according to 10x 5' immune profiling.  Single-cell 10 VDJ libraries were constructed from the isolated BM plasma cells following the demonstrated protocol Direct target enrichment Chromium Single Cell V(D)J Reagent Kits (CG000166 REV A). Briefly, single cells were co-encapsulated with gel beads (10 Genomics, 1000006) in droplets using three lanes of one Chromium Single-Cell A Chip (10 Genomics, 1000009) with a target loading of 13,000 cells per reaction. V(D)J library construction was carried out using the Chromium Single Cell 5 Library Kit (10 Genomics, 1000006) and the Chromium Single Cell V(D)J Enrichment Kit, Mouse B Cell (10 Genomics, 1000072).
Experimental Factor: infect
Lymphocytic choriomeningitis mammarenavirus
Experimental Factor: stimulus
none
Experimental Factor: protocol
10x Ig enrichment