GSM8425096: HDM-specific, anti-IL4Ra-treated, Plasma cells,  Bone Marrow, biol. repl. 6, scRNAseq; Mus musculus; RNA-Seq
The IgE-secretion trap method and single cell preparation is described in detail in our recent work. In brief, the IgE-secretion trap was performed with an StAv-ectoFcepsilon to specifically capture IgE-secreting plasma cells. Cells were sorted and submitted for scRNA-seq gene expression. We sequenced PCs from dLN and BM of mice exposed to HDM for 15 weeks, and from BM of mice exposed to HDM for 19 weeks with the last 8 weeks with anti-IL4Ra. At the end of the experiment, bone marrow and lung draining lymph nodes were collected and processed. Draining lymph nodes were mashed on 12-well, 70-mm filter plates (Corning Costar) in RPMI 1640 media to generate single cell suspensions. For BM extraction, femurs were cut at both ends, placed in a PCR plate with holes punched at the bottom, and spun down for 3 min at 500g. RBC lysis was performed on single cell suspensions from draining lymph nodes and bone marrow. B cells and plasma cells were isolated using EasySep mouse pan-B cell isolation kit (STEMCELL, cat: 19844) by negative selection to remove unwanted cells (CD4, CD8, CD11c, CD49b, CD90.2, Ly-6C/G (Gr-1), TER119). Purified cells were processed and secretion trap was assembled as described in the previous paragraph. Secreted IgE was captured using StAv-ectoFceRIa for 1 h at 37C with shaking at 400 rpm. Following IgE secretion trap, cells were washed and stained with an antibody mix containing B220 (RA3-6B2), CD138 (281-2), IgG1 (A85-1), IgE (R35-72), DUMP (IgM (II/41), IgD (11-26c.2a), TCRb (H57-597), Ly6G (1A8-Ly6g), CD49b (DX5), and CD11b (M1/70)) along with barcoded antigens (Der p1, Der p2, Der f1, and Ole e1). Finally, cells were labeled with DAPI before they were sorted on a BD Symphony S6. Human allergic donor BM and matching donor sera were obtained from StemCell Technologies. Allergy status of each donor sera was checked by ImmunoCAP test (Phadia, Thermo Fisher Scientific) following the manufacturer's instructions. To obtain mononuclear cells fresh BM aspirate was diluted 7:1 with PBS and 2mM EDTA and loaded onto 15mL of Ficoll in SepMate tube (Stem Cell Technologies). Samples were spun in SepMate tubes at 1200g for 20 minutes. Top layer of cells was removed, and cells were washed twice with autoMacs rinsing solution at 300g for 8 minutes (StemCell Technologies). B and antibody secreting cells were isolated using magnetic bead depletion (EasySep Human Pan-B Cell Isolation Kit, StemCell Technologies, Inc., Vancouver, CA, Cat: 19554) from single cell suspensions of allergic BM cells according to manufacturer's protocol. Library was performed according to the manufacter's instructions (single cell 5' v2 protocol, 10x Genomics). Cells captured by secretion trap were suspended in PBS with 0.04% w/v BSA and loaded onto a Chromium® Controller device (10x Genomics) at 15,000 cells per lane. Partitions of the cells in barcoded beads were formed and the cells lysed. Reverse transcription was performed followed by breakdown of the partition. RNA-seq, Feature Barcode, and V(D)J libraries were prepared using a Chromium® Next GEM Single Cell 5' Kit, v.2 (10x Genomics). After amplification of the libraries, cDNA was split into separate RNA-seq, Feature Barcode, and V(D)J aliquots. To enrich the V(D)J aliquot for BCR sequences, the Chromium® Automated Single Cell Mouse and Human BCR Amplification & Library Construction Kit (10x Genomics) was used. Feature Barcode libraries for DNA-barcoded Antigens or DNA-barcoded cell surface proteins were prepared using a Chromium® 5' Feature Barcode Library Construction kit (10x Genomics). Paired-end sequencing was performed on an Illumina NovaSeq® 6000 sequencing system for RNA-seq, feature barcode, and V(D)J libraries. For RNA-seq and Feature Barcode libraries, the Cell Ranger Single-Cell Software Suite, version 6.1.1 (10x Genomics) was used to perform sample de-multiplexing, alignment, filtering, and UMI counting.