BioProject

Normalization of RNA sequencing (RNA-seq) data for gene expression comparison is essential to ensure accurate gene expression quantification. More...

Normalization of RNA sequencing (RNA-seq) data for gene expression comparison is essential to ensure accurate gene expression quantification. It has been argued that samples with large differences in global expression level cannot be properly normalized without spike-in control RNAs, however, spike-in controls are expensive and not yet widely used. Here, we presented a spike-in independent quantitative RNA sequencing (siqRNA-seq) method, which uses reads from genome DNA as an internal reference to quantify gene expression level. We showed that siqRNA-seq profiles gene expression as traditional RNA-seq, but allows to identify different expression genes between samples with distinct mRNA content. We also showed siqRNA-seq enable us to assess the copy number of mRNA per cell without counting cells and adding spike-ins. Thus, siqRNA-seq provides a convenient and versatile means to quantitatively profile the mRNA landscape in cells. Overall design: To develop a spike-in-independent method for gene expression quantification, we designed siqRNA-seq that utilize genome DNA (gDNA) as internal reference for normalization . As we known, each cell has a set of genomic DNA, so we considered that it can be taken as an internal reference but with large molecular weight. To that end, whole nucleic acids were extracted from samples to construct two libraries in parallel for sequencing in siqRNA-seq, mRNA library and mRNA&gDNA library , respectively. Genome DNA was removed by digested with DNase I in mRNA library while retained in mRNA&gDNA library as internal reference. mRNA was reverse-transcribed with oligo(dT) as the primer to synthesis the first strand of the complementary DNA (cDNA). To reduce bias and simplify our pipeline, fragmented cDNA without second strand synthesis and gDNA were denatured by heat following library preparation using a high-efficient ssDNA ligation technique, Adaptase from Accel-NGS 1S Plus DNA Library Kit (Swift Accel-NGS). Adaptase is a commercial enzyme mixture with very high ligation efficiency and low bias for single-stranded DNA (ssDNA) library construction with strand-specific information, which we have used to develop ssDRIP-seq for R-loop profiling and DEtail-seq for DNA break detection . Compared to traditional RNA-seq, mRNA library in siqRNA-seq was directly prepared from single-stranded cDNA, therefore we called this method as ssRNA-seq hereafter. Less...