Schematic of the experimental design to assess the variability of spike-in addition in a plate-based scRNA-seq protocol. (A) A cell is sorted into each well of a plate and lysed. For one set of wells, an equal volume of each spike-in set is added separately, along with the reverse transcription (RT) reagents. For another set of wells, an equal volume of a pooled mixture of the two spike-ins is added into each well (done twice to keep the protocol consistent). Reverse transcription, PCR amplification, library generation, and sequencing were then performed. (B) The log2-ratio between the total counts of the two spike-in sets was computed for each well. The variance of the log-ratio was estimated from all wells with separate addition of spike-ins and from wells with addition of the premixed pool. The difference between these two estimates represents the variance attributable to volume addition.
