GSM6637190: MemoryB  B.1.1.529 SARS-CoV-2; Mus musculus; RNA-Seq
Memory B-cell fractions were enriched by magnetic positive selection according to the manufacturer's protocol (Miltenyi Biotec, 130-095-838). Briefly, spleen and lymph nodes were gently homogenized and red blood cells were lysed in ACK lysis buffer (Lonza). The remaining cells were washed by PBS with 2%FBS. Thereafter, B cells were labeled with memory B cell biotin-antibody cocktail combined with anti-biotin microbeads. The enriched memory B cells were eluted and mixed with 25ug of Omicron RBD recombinant protein or 25ug of recombinant CD22 extracellular domain with his tag and incubated for 30mins on ice. After incubation, the complex was washed and respectively incubated with anti-his-APC antibody and anti-APC microbeads. The final antigen-specific B cells were eluted in PBS and stored on ice until use. For GPRC5D, the memory B cells were labeled with APC anti-mouse IgG1 and anti-mouse IgG2ab and incubated for 30mins on ice. After incubation, the complex was washed and incubated with anti-APC microbeads. The final IgG1 or IgG2ab positive B cells were eluted in PBS and stored on ice until use. Plasma B cells were collected by fragmenting and rinsing bone marrows with PBS containing 2% FBS. Non-plasma cells were labeled with a biotin-conjugated antibody cocktail combined with anti-biotin microbeads and separated using a magnetic rack according to the manufacturer's protocol (Miltenyi Biotec, 130-092-530). Purified plasma B cells were eluted and sequentially incubated with CD138 microbeads for an additional 15 minutes at 4℃. The final CD138+ plasma B cells were eluted in PBS and stored on ice until use. PBMC cells were isolated from fresh whole blood by using SepMate-15 tubes with Lymphoprep gradient medium (StemCell Technologies) after centrifugation at 1200g for 20 minutes. Poured top layer solution that contained PBMCs from SepMate tubes to a new falcon tube and washed once with PBS+2%FBS, resuspended with PBS and stored on ice until use. Cell suspension were normalized to 1000 cells/ml and loaded to achieve targeted cell recovery to 10000 cells. The samples were subjected to 14 cycles of cDNA amplification. Following this, BCR-enriched libraries were prepared according to the manufacturer's protocol (10x Genomics). All libraries were sequenced using a Miseq (Illumina) with 26X91 read length. scVDJ-seq