Extended Data Fig. 1: Performance and quality-control-related analyses for sci-RNA-seq3.

a, Comparison of fixation conditions in human HEK-293T cells. PFA-fixed nuclei yielded the highest numbers of UMIs. Cell number: n = 21 for fresh nuclei, 17 for frozen nuclei, 32 for PFA-fixed cells and 31 for PFA-fixed nuclei. b, Tn5 transposomes loaded only with N7 adaptor (cell number, n = 13 cells) increased UMI counts by over 50%, relative to the standard Nextera Tn5 (cell number, n = 11), in human HEK-293T cells. c, Bar plot showing the number of reverse transcription wells used for each of 61 mouse embryos. d, Histogram showing the distribution of raw sequencing reads from each PCR well in sci-RNA-seq3. e, Scatter plot of mouse (NIH/3T3) versus human (HEK-293T) UMI counts per cell. f, g, Box plot showing the number of UMIs and purity (proportion of reads mapping to the expected species) per cell from HEK-293T (cell number n = 7,943) and NIH/3T3 cells (cell number, n = 10,914). At a sequencing depth of 23,207 reads per cell, we observed a median of 5,461 UMIs per HEK-293T cell and 5,087 UMIs per NIH/3T3 cell, with 3.9% and 2.9% of reads per cell mapping to incorrect species, respectively. h, Box plot comparing the number of UMIs per cell (downsampled to 20,000 raw reads per cell) for sci-RNA-seq3 (cell number, n = 689 for HEK-293T and 997 for NIH/3T3) versus sci-RNA-seq (cell number, n = 47 for HEK-293T and 120 for NIH/3T3). i, Correlation (Pearson’s correlation) between gene expression measurements in aggregated profiles of HEK-293T from sci-RNA-seq3 nuclei versus sci-RNA-seq cells. j, Scatter plot showing correlation between number of reverse transcription wells used and number of cells recovered per embryo. k, Box plot showing the number of genes and UMIs detected per cell. l, Box plot showing the number of UMIs detected per cell from embryos across five developmental stages. Cell number: n = 152,120 for E9.5; 378,427 for E10.5; 615,908 for E11.5; 475,047 for E12.5; 437,150 for E13.5. m, Histogram showing the distribution of the cell doublet score for the actual mouse embryo data versus doublets stimulated by Scrublet. n, Scatter plot of the number of cells profiled per reverse transcription well and the detected doublet-cell ratio. Blue line shows the linear regression. The detected doublet-cell rate was modestly correlated with number of cells profiled per well during reverse transcription (Spearman’s ρ = 0.35). o, Scatter plot of unique reads aligning to Xist (female-specific) versus chrY transcripts (male-specific) per mouse embryo. Sex assignments of individual embryos inferred from these data. p, Bar plot showing the number of male and female embryos profiled at each developmental stage. q, t-SNE of the aggregated transcriptomes of single cells derived from each of 61 mouse embryos results in 5 tightly clustered groups perfectly matching their developmental stages (embryo number, n = 61). r, Pseudotime trajectory of pseudobulk RNA-seq profiles of mouse embryos (embryo number, n = 61); identical to Fig. 1c, but coloured by pseudotime. s, The E10.5 embryos were ordered by pseudotime. The 3 earliest versus 3 latest (in pseudotime) E10.5 embryos are shown in photographs, and appear to potentially be morphologically distinct. Notably, the distinct colouring of E10.5 embryos positioned earlier versus later in developmental pseudotime is potentially due to different levels of haemoglobin. For all box plots: thick horizontal lines, medians; upper and lower box edges, first and third quartiles, respectively; whiskers, 1.5 times the interquartile range; circles, outliers.