Isolation and Sequencing of SARS-CoV-2 Specific B Cells

Peripheral blood samples from convalescent COVID-19 subjects were obtained with informed consent under the approval of the University of Washington Institutional Review Board (Gale Lab; 00009810) and isolated as previously described (Rodda et al., 2021 (link)). Briefly, peripheral blood mononuclear cells were thawed, washed, and stained with a decoy tetramer and then RBD tetramer; bound cells were magnetically enriched (Miltenyi Biotec) and stained with surface antibodies. Single tetramer-positive B cells were index-sorted using a FACS Aria II and collected in 96-well plates containing SMART-Seq v4 capture buffer (Takara Bio) for BCR sequencing.

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Hale M., Netland J., Chen Y., Thouvenel C.D., Smith K.N., Rich L.M., Vanderwall E.R., Miranda M.C., Eggenberger J., Hao L., Watson M.J., Mundorff C.C., Rodda L.B., King N.P., Guttman M., Gale M J.r., Abraham J., Debley J.S., Pepper M, & Rawlings D.J. (2022). IgM antibodies derived from memory B cells are potent cross-variant neutralizers of SARS-CoV-2. The Journal of Experimental Medicine, 219(9), e20220849.

Publication 2022

FACS Aria II SMART-Seq v4 capture buffer

Aria B cells Blood Buffer Cells Covid 19 Gale Institutional review board Peripheral blood mononuclear cells Surface antibodies Tetramer

Corresponding Organization : University of Washington

Other organizations : Boston VA Research Institute, Harvard University

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Variable analysis

independent variables

Peripheral blood samples from convalescent COVID-19 subjects

dependent variables

Tetramer-positive B cells

control variables

Peripheral blood mononuclear cells were thawed, washed, and stained with a decoy tetramer and then RBD tetramer
Bound cells were magnetically enriched (Miltenyi Biotec) and stained with surface antibodies
Single tetramer-positive B cells were index-sorted using a FACS Aria II and collected in 96-well plates containing SMART-Seq v4 capture buffer (Takara Bio) for BCR sequencing

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