Figure 5
Freezing samples at an early stopping point during RevGel-seq protocol does not modify scRNA preparation performances. (A) Schematic representation of the timing differences between the “Control” and “Early stop” samples (see “Methods” for specific steps). “Control” samples were obtained by freezing the RT reaction mixture at − 20 °C overnight, while “Early stop 80 °C” and “Early stop dry ice” samples were obtained by freezing (either at − 80 °C or in dry ice, respectively) immediately after applying lysis buffer to the hydrogel. The RevGel-seq protocol was resumed on a subsequent day for all samples. (B) Comparison of performance indicators between the tested conditions (average of 3 PCR tubes for each condition). Gene and transcript counts per analyzed cell at 50,000 raw reads per cell with raw read downsampling. (C,D) Projection of scRNA-seq data on 2D (UMAP) of the pooled samples (pool of data from 1 PCR tube for all 3 conditions), shown per condition (C) and per determined cell species (D). (“undefined” corresponds to cells with a cell barcode purity lower than 95%; see Methods section “Pre-processing pipeline”, iii). With respect to cell capture yields, obtained cell species proportions were as follows: human 45.8%, mouse 49.9%, undefined 4.3% (Control sample); human 48.6%, mouse 52.5%, undefined 3.9% (Early stop dry ice); and human 46.2%, mouse 48.4%, undefined 5.4% (Early stop − 80 °C).
