Sample Detail

DescriptionProtocols: Splenic B cells were isolated from mice aged 9-16wks by depletion of CD43+ cells 65 using magnetic microbeads (Miltenyi Biotec, Cat no. 130-049-801). Purity of the isolated B cells was assessed using anti-B220 antibody (clone RA3-6B2) and found to be >98% for B220+ cells. To induce class-switching to IgG1 and IgE, B cells were seeded into 96-well round bottom plates at a density of 105 cells/200 μl RPMI supplemented with 10% FBS, 50 μM 2-mercaptoethanol, 100 U/ml penicillin- 100 μg/ml streptomycin (Invitrogen; Cat no. 15140122), 25 ng/ml recombinant mouse IL-4 (R&D Systems; Cat no. 404-ML), 40 μg/ml LPS (Sigma-Aldrich; L4391 or L3129) and 20 ng/ml recombinant mouse BAFF of (R&DSystems; Cat no. 2106-BF). Cells were harvested after 24-hours for FACS as detailed in the cell-lysis protocol\. After treatment as described in the stimulation protocol, single cells were deposited into 96-well PCR plates containing lysis buffer using an iCyt Synergy cell sorter. Plates were then immediately spun down and stored at -80°C until further processing. The generation of the single-cell libraries followed the Smart-seq2 protocol42, then switched to the Fluidigm C1 protocol for tagmentation and purification of the pooled libraries (Fluidigm; PN 100-7168). The pooled, barcoded libraries were quantified by qPCR and checked for size distribution using an Agilent Bioanalyzer 2100.

Organism Info

Taxon ID10090
Common Name
Scientific NameMus musculus
Anonymized Name
Individual Name