Figure S3. CRISPR/Cas9 screen for... | Rockefeller University Press

Figure S3.

$CRISPR/Cas9 screen for identifying new regulators of the TD B cell responses. (A) Schematic diagram of the in vivo CRISPR/Cas9 screening experiments used to study the TD B cell responses. Naïve B cells isolated from donor mice were transduced with LV particles, each expressing one of the 882 sgRNAs and the mCherry reporter protein. After infection, B cells were cultured in vitro for 3 days in the presence of OP9 cells and sBAFF. Transduced mCherry+ donor B cells were sorted by flow cytometry and transferred to recipient mice, which were immunized 16 h later with NP-KLH/alum. 7 days after immunization, splenic mCherry+ CD45.2+ cells were enriched by anti-CD45.1 antibody–mediated MACS depletion of the CD45.1+CD45.2+ recipient cells. Plasmablasts and GC B cells were isolated from the enriched fraction by flow cytometric sorting, followed by DNA extraction, library preparation, and DNA sequencing. DNA was also extracted from a fraction of the sorted transduced mCherry+ donor B cells before immunization, followed by library preparation and DNA sequencing. (B) Flow cytometric analysis of the enriched mCherry+ CD45.2+ B cells from recipient mice. The gates used for the sorting of plasmablasts (TACI+CD138+) and GC B cells (CD19+GL7+CD95+) are indicated. The percentages of mCherry+ plasmablasts and mCherry+ GC B cells within the sorted cell population are shown. (C) Numbers of sorted plasmablasts and GC B cells (left), the percentage of mCherry+ plasmablasts and mCherry+ GC B cells within the sorted cell population (middle), and the numbers of mCherry+ plasmablasts and mCherry+ GC B cells (right) were determined for individual recipient mice analyzed in two independent screening experiments. (D) Total sgRNA representation in each plasmablast and GC B cell replicate sample analyzed in two screening experiments. The sgRNA representation was estimated by dividing the number of mCherry+ plasmablasts or GC B cells by the number of the 882 sgRNAs constituting the sgRNA library. Each plasmablast and GC B cell replicate sample contained cells that were obtained from five or six recipient mice after pooling. Each colored box represents the individual contribution of each mouse to the sgRNA representation of the entire replicate sample.$

CRISPR/Cas9 screen for identifying new regulators of the TD B cell responses. (A) Schematic diagram of the in vivo CRISPR/Cas9 screening experiments used to study the TD B cell responses. Naïve B cells isolated from donor mice were transduced with LV particles, each expressing one of the 882 sgRNAs and the mCherry reporter protein. After infection, B cells were cultured in vitro for 3 days in the presence of OP9 cells and sBAFF. Transduced mCherry⁺ donor B cells were sorted by flow cytometry and transferred to recipient mice, which were immunized 16 h later with NP-KLH/alum. 7 days after immunization, splenic mCherry⁺ CD45.2⁺ cells were enriched by anti-CD45.1 antibody–mediated MACS depletion of the CD45.1⁺CD45.2⁺ recipient cells. Plasmablasts and GC B cells were isolated from the enriched fraction by flow cytometric sorting, followed by DNA extraction, library preparation, and DNA sequencing. DNA was also extracted from a fraction of the sorted transduced mCherry⁺ donor B cells before immunization, followed by library preparation and DNA sequencing. (B) Flow cytometric analysis of the enriched mCherry⁺ CD45.2⁺ B cells from recipient mice. The gates used for the sorting of plasmablasts (TACI⁺CD138⁺) and GC B cells (CD19⁺GL7⁺CD95⁺) are indicated. The percentages of mCherry⁺ plasmablasts and mCherry⁺ GC B cells within the sorted cell population are shown. (C) Numbers of sorted plasmablasts and GC B cells (left), the percentage of mCherry⁺ plasmablasts and mCherry⁺ GC B cells within the sorted cell population (middle), and the numbers of mCherry⁺ plasmablasts and mCherry⁺ GC B cells (right) were determined for individual recipient mice analyzed in two independent screening experiments. (D) Total sgRNA representation in each plasmablast and GC B cell replicate sample analyzed in two screening experiments. The sgRNA representation was estimated by dividing the number of mCherry⁺ plasmablasts or GC B cells by the number of the 882 sgRNAs constituting the sgRNA library. Each plasmablast and GC B cell replicate sample contained cells that were obtained from five or six recipient mice after pooling. Each colored box represents the individual contribution of each mouse to the sgRNA representation of the entire replicate sample.