Extended Data Fig. 3: Evaluating HiMAP-seq for sensitivity and accuracy.

a, Distribution of detected gene counts within samples of MDA-MB-231 cells, each comprising varying cell numbers (n = 16 for each group). Sequencing data from pooled samples amounted to 16,221 Mbp, with genes classified as ‘detected’ if their read count in the sample exceeded 0. The average number of detected genes within each group was computed based on data collected from the 16 samples. b, Distribution of detected gene counts in samples of MDA-MB-231 cells with varying sequencing depths. Reads from the MDA-MB-231 cells sample containing 3,500 cells were randomly selected, and the number of detectable genes was recorded at various read depths. c and d, Comparison of HiMAP-seq with RNA-seq. Heat maps displaying the log₂(fold change) of genes in MDA-MB-231 cells (c) and HepG2 cells (d) both treated with 10 μM JQ1. The log₂(fold change) values were determined using either HiMAP-seq or RNA-seq. e and f, Comparison of HiMAP-seq with qPCR. Heat maps displaying the log₂(fold change) of genes in MDA-MB-231 cells (e) and HEK293T cells (f) both treated with 10 μM JQ1. The log₂(fold change) values were detected using HiMAP-seq or qPCR. g and h, The Pearson correlation (two-sided) between DMSO and JQ1 treated samples. For MDA-MB-231 cells, the treatment concentrations were 10 μM and 20 μM, respectively (g). The boxplot displays the Pearson correlation between identically perturbed samples and the correlation between two random samples. Self-samples represent biological replicate samples. Box plots show the median (center line), 25th and 75th percentiles (box bounds), and full data range. From left to right, the medians are: 0.94, 0.95, 0.97, 0.97, 0.96, 0.96, 0.96, and 0.97, respectively. The interquartile ranges are: 0.94-0.95, 0.95-0.96, 0.96-0.97, 0.967-0.970, 0.94-0.96, 0.96-0.97, 0.96-0.97, and 0.96-0.97, respectively. The minimum and maximum values are: 0.84-0.97, 0.91-0.97, 0.89-0.97, 0.95-0.97, 0.91-0.97, 0.95-0.97, 0.89-0.97, and 0.93-0.98, respectively. Sample sizes (n) are: 5,538, 1,846, 5,289, 1,763, 5,463, 1,821, 5,259, and 1,753, respectively (h). Comparisons were made using Student’s t-test. i, Comparison of HiMAP-seq with RNA-seq from public datasets. Pearson correlation coefficients (two-sided) were calculated to evaluate mRNA quantification consistency between RNA-seq and HiMAP-seq in MDA-MB-231 cells. For HiMAP-seq and RNA-seq (GSE236250) samples, the perturbation concentration was 20 μM and 25 μM, respectively. Genes with |fold change | ≥ 1.5 were included in this analysis, and the results are presented as log₂(fold change).