title: GSM7151552: Sp, Fo_pos, 4h stim, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Total splenocytes from sIgM-/-IL10GFP mouse were isolated by mechanical disruption through a 70um filter, red blood cells were lysed, and individual live cells counted. Cells were were stimulated at a concentration of 4x10^6/ml with 10 ng/ml PMA, 0.5 ug/ml Iono, and 10 ug/ml LPS for 4-h at 37C, 5% CO2, 13% O2. At end of stimulation, B cells were enriched using CD19 microbeads (Miltenyi 130-121-301) following the manufacturer's protocol then stained for B cell subset markers and FACS sorted on BD FACSAriaII for IL-10+ and IL-10- B1a, Mz, and Fo B cells subsets. Positive GFP expression (IL-10) was based on a GFP negative wildtype mouse control. Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, sorted B cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent. Full-length V(D)J segments (10X barcoded) were enriched from amplified cDNA via PCR amplification with primers specific to Ig constant regions.  During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.