Single cell RNA-seq of 10 ug/ml anti-IgM-stimulated mouse B cell: adult spleen
Splenic B cells were positively sorted by staining with anti-B220-APC (Biolegend, San Diego, CA). The sorted splenic B cells were treated with different concentrations of anti-IgM (1 hour, 0, 0.01, 0.1, 1, 10 μg/ml) and used for single cell RNA-seq. All of the single cell RNA-seq samples were quantified and evaluated using the MultiNA DNA-12000 kit (Shimadzu) with a modified sample mixing ratio (1:1:1; sample, marker and nuclease-free water) in a total of 6 μL. The length and yield of the library DNA were calculated in the range of 150-3000 bp. The library DNA yield in particular was estimated as 0.5-fold the value quantified from the modified MultiNA condition. Subsequently, we pooled each 200 fmol of library DNA in each well of a 96-well plate. The pooled library DNA was evaluated based on the averaged length and concentration using the Bioanalyzer Agilent High-Sensitivity DNA Kit in the range of 150-3000 bp and the library quantification kit (Kapa Biosystems). Finally, 1.5 pM pooled library DNA was sequenced using HiSeq2500 (Illumina) as paired end.