A method for simultaneous detection of small and long RNA biotypes by ribodepleted RNA-Seq

Abstract

RNA sequencing offers unprecedented access to the transcriptome. Key to this is the identification and quantification of many different species of RNA from the same sample at the same time. In this study we describe a novel protocol for simultaneous detection of coding and non-coding transcripts using modifications to the Ion Total RNA-Seq kit v2 protocol, with integration of QIASeq FastSelect rRNA removal kit. We report highly consistent sequencing libraries can be produced from both frozen high integrity mouse hippocampal tissue and the more challenging post-mortem human tissue. Removal of rRNA using FastSelect was extremely efficient, resulting in less than 1.5% rRNA content in the final library. We identified > 30,000 unique transcripts from all samples, including protein-coding genes and many species of non-coding RNA, in biologically-relevant proportions. Furthermore, the normalized sequencing read count for select genes significantly negatively correlated with Ct values from qRT-PCR analysis from the same samples. These results indicate that this protocol accurately and consistently identifies and quantifies a wide variety of transcripts simultaneously. The highly efficient rRNA depletion, coupled with minimized sample handling and without complicated and high-loss size selection protocols, makes this protocol useful to researchers wishing to investigate whole transcriptomes.

Figure 1

An overview of the protocol described here for simultaneous detection of coding and non-coding RNA by RNA-Seq. Created in BioRender.com.

Figure 2

(a–c) Representative Bioanalyzer electropherograms of (a) total input RNA, (b) RNA after 8 min fragmentation by RNase III, and (c) final amplified cDNA library from mouse hippocampal RNA. (d–f) Representative Bioanalyzer electropherograms of (d) total input RNA, (e) RNA after 1 min fragmentation by RNase III, and (f) final amplified cDNA library from human MTG RNA. (g–i) Representative Bioanalyzer electropherograms of unamplified cDNA produced from adapter ligation for (g) 30 min at 30 °C (h) 60 min at 30 °C, and (i) 16 h at 16 °C. Yield of cDNA (size 50 to 1000 bp) markedly increases from 53 pg/μL (g) to 528 pg/μL (h) to 745.5 pg/μL (i) with increasing ligation time. (j) RNA reads mapped to the ENSEMBL Mus musculus GRCm38.95 annotated genome and (k) to the ENSEMBL Homo sapiens GRCh38.96 annotated genome. Uniquely mapped reads, multi-mapped reads, reads mapped to too many loci (> 10), and unmapped reads for each sample shown as a percentage of total trimmed and filtered reads.

Figure 3

Breakdown of number of genes identified by RNA biotype. (a) Number of genes detected at > 1 Reads per Kilobase Million (RPKM) and > 0.1 RPKM for each mouse sample and human sample, divided by gene biotype. RPKM here was used as a proxy for normalized expression. (b,c) Percentage RNA reads mapped to gene biotypes for (b) mouse and (c) human samples, averaged across samples.

Figure 4

Box and whisker plot showing the range of percentages of reads mapped to gene biotypes for (a) mouse and (b) human samples. The majority of RNA species in both samples show very small ranges, while some (notably snRNA and snoRNA) are more variable between samples.

Figure 5

Correlation between Ct values from qPCR and normalized sequencing reads mapped to select genes (a) and miRNA (b). Gene sequencing reads were normalized for library size and gene length using RPKM, while miRNA reads were normalized for library size using CPM. Both sets of data passed Shapiro–Wilk tests of normality (p > 0.05). The linear regression line, confidence interval, Pearson’s correlation coefficient, and significance value are indicated.