Fig. 1: Research design and general effect of DSN-based cDNA-normalized PacBio RNA-seq.

a Overall design of the research. Generally, cDNA generated from human peripheral blood, two SRCCs, and their adjacent non-malignant gastric mucosa samples (as listed in the white box) were normalized by DSN digestion and non-normalized, followed by PacBio RS II SMS. The effect was compared between normalization and non-normalization, mainly in respects of the four items as indicated in the gray box. Each original cDNA sample was also sequenced using Illumina SGS platforms for assisting data analysis. The SGS and normalized SMS data of SRCCs and paired samples were also used for SRCC transcriptome profiling and comparison (shown in purple). b General experimental and data analytic procedure of cDNA-normalized SMS. The step in the box with dash lines was optional. c The principle of DSN-based cDNA normalization (referring to Zhulidov et al.¹³). Basically, DSN specifically digests the incompletely hybridized double-strand (ds) cDNA molecules, which mostly represent the genes with high expression, and eventually, the transcripts of genes with uneven expression originally turn equal. d–e General effect of cDNA normalization in reducing the highly expressed genes and enriching the lowly expressed genes in human blood samples. d The representative genes with varied original expression (quantified by SGS, left gray bars) were quantified with the number of ROIs mapped to the corresponding genes (normalized as per 100k total ROIs) in the normalized or non-normalized SMS libraries. e Expression analysis of ERCC genes with real-time quantitative PCR. The graph represents the mRNA expression levels of indicated genes that were normalized to the Ct values of ERCC-00076. The experiments were performed in triplicates. “Standard” showed the actual ratio of molecular concentrations in the original reagent.