NextSeq 500 sequencing; Single cell RNA-seq of hemagglutinin specific B cells after influenza virus infection: VDJ data
Single cell RNA-seq of hemagglutinin specific B cells after influenza virus infection: VDJ data
C57BL/6 mice were infected with PR8 H1N1 virus and were euthanized on different days post- infection. Organs such as lungs, spleen and mediastinal lymph nodes (mln) were isolated. The same organs from naïve mice were used as controls. Spleen and mln were mashed and passed through a 70µm filter to obtain single cell suspension. Lungs were processed into single cell suspension using the mouse lung dissociation kit (Miltenyi Biotec) according to manufacturer's instruction. Splenocytes and lung cells were enriched for total B cells using the EasySep Mouse Pan-B Cell Isolation kit (Stemcell Technologies) while whole mln cells were used for downstream processing. The cells were incubated for one hour at 4°C with a cocktail of fluorochrome-labelled antibodies consisting of anti-CD3-BV510 (cat. no: 563024, BD Biosciences), anti-B220-APC-Cy7 (cat. no: 552094, BD Biosciences) and anti-IgD-Pacific Blue (cat. no: 405712, BD Biosciences), and 1µg/ml biotinylated recombinant hemagglutinin (rHA) (Whittle et al., 2014) conjugated to streptavidin APC (cat. no: S868, Invitrogen). To exclude dead cells, the cells were washed and stained with LIVE/DEAD™ Fixable Aqua Dead Cell Stain (cat. no: L34957, Invitrogen) according to manufacturer's instruction.  Mice were anesthetized with isoflurane and infected through nasal inoculation with 50 TCID50 Influenza A/Puerto Rico/8/34 (PR8) (Molecular clone; H1N1) diluted in HBBS containing 0.1% BSA. A maximum of 10,000 live HA-specific mature B cells (CD3-B220+IgD-rHA+) were sorted and collected in a BD FACSAria fusion or BD FACSAria III (BD Biosciences) cell sorter. Between 1500-10,000 sorted HA-specific mature B cells from individual organs were processed into single cells in a chromium controller (10X genomics) Libraries were generated following standard10x protocol . After amplification of the cDNA, 5´gene expression library and enriched B cell library, with paired heavy and light chain were generated  from cDNA of the same cell using Chromium single cell VDJ reagent kit (V1.1 chemistry, 10X genomics).