NextSeq 500 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
For protein immunizations, a 6-week-old male C57BL/6 mouse was repeatedly immunized five times every other week s.c. into the flank with 10 g of human TNFR2 protein (Peprotech, 31012)/20 g MPLA (Sigma, L6895) adjuvant and sacrificed 1 week afterwards. Likewise, a 6-week-old female BALB/c mouse was injected three times with 4 and 3 weeks intervals s.c. into the flank with 100 g of OVA (Sigma, A5503)/20 g MPLA (Sigma, L6895) adjuvant and sacrificed 2 weeks after the final boost. 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
none
Experimental Factor: stimulus
ovalbumin
Experimental Factor: protocol
10x Ig enrichment