Prime-seq, efficient and powerful bulk RNA-sequencing

Abstract

With the advent of Next Generation Sequencing, RNA-sequencing (RNA-seq) has become the major method for quantitative gene expression analysis. Reducing library costs by early barcoding has propelled single-cell RNA-seq, but has not yet caught on for bulk RNA-seq. Here, we optimized and validated a bulk RNA-seq method we call prime-seq. We show that with respect to library complexity, measurement accuracy, and statistical power it performs equivalent to TruSeq, a standard bulk RNA-seq method, but is four-fold more cost-efficient due to almost 50-fold cheaper library costs. We also validate a direct RNA isolation step that further improves cost and time-efficiency, show that intronic reads are derived from RNA, validate that prime-seq performs optimal with only 1,000 cells as input, and calculate that prime-seq is the most cost-efficient bulk RNA-seq method currently available. We discuss why many labs would profit from a cost-efficient early barcoding RNA-seq protocol and argue that prime-seq is well suited for setting up such a protocol as it is well validated, well documented, and requires no specialized equipment.

Early nucleocapsid-specific T cell responses associate with control of SARS-CoV-2 in the upper airways and reduced systemic inflammation before seroconversion

“…RNA isolation and sequencing was performed as described previously (Pekayvaz et al, 2022). Briefly, libraries were prepared from immune cell subsets (n = 500 cells each) using the Prime-seq protocol 55 , and quality was determined using a High Sensitivity DNA Kit (Agilent Bioanalyzer). Paired-end sequencing (150 bp) was performed using an S1 or an S4 flow cell on a NovaSeq System (Illumina).…”

The EBNA2-EBF1 complex promotes oncogenic MYC expression levels and metabolic processes required for cell cycle progression of Epstein-Barr virus-infected B cells

“…Total cellular RNA was isolated on individual days and high quality cDNA libraries for RNAsequencing analyses were generated (Figure S 4) using the bulk RNA-sequencing method primeseq (Janjic et al, 2021). Principal component analysis (PCA) of RNA sequencing results for protein coding genes confirmed the considerable switch of gene expression patterns caused by viral infection of both viruses (Figure 5B).…”

“…Random hexamers were used as primers. A reaction without reverse transcriptase was used as a negative RNA and cDNA library preparation for RNA-sequencing RNA sequencing was performed using the prime-seq method based on the scRNA-seq method SCRB-seq (Bagnoli et al, 2018;Janjic et al, 2021). 10,000 cells per sample were sorted into a 96 well plate containing 50 µl RLT plus Lysis Buffer (Qiagen) supplemented with 1% 2mercaptoethanol and frozen at -80 °C immediately.…”

UHMK1 is a novel splicing regulatory kinase

“…Splicing analysis of transcriptome data revealed that UHMK1 modulation affected over 200 alternative splicing events, most of which led to exon inclusion/exclusion and produced alternative isoforms. Since our analysis was performed on Prime-seq data [32] and the reads were sequenced mainly from the 3' end of the transcripts, one limitation of our analysis is that we only evaluated alternative splicing events occurring within this region of the transcripts. Thus, we cannot consider it a global splicing analysis as the identified ASEs are likely to be underrepresented.…”

“…Replicates were harvested in 3 and 6 consecutive days, for UHMK1 knockdown and overexpression, respectively. A bulk RNA barcoding and sequencing protocol, named Prime-seq [32] was used for library preparation. Briefly, RNA was cleaned up using solid phase reversible immobilization (SPRI) beads (Sera Mag™, GE Healthcare Life Sciences, Chicago, IL, USA) in a homemade bead binding buffer containing 22% polyethylene glycol (PEG).…”