Optimization of rRNA Depletion by Testing Various Kits

RNA sequencing (RNAseq) can be used to compare gene expression between different groups, identify mutations, and confirm gene knockouts. In RNAseq, RNA is extracted and built into a sequenceable library to analyze the transcriptome. Ribosomal RNA (rRNA) makes up roughly 90% of the total RNA extracted and does not provide useful transcriptome information, thus it must be removed in order to perform transcriptional analysis. At MBP Titan, the protocol used KAPA’s RiboErase Depletion kit using custom methanotroph oligonucleotides to specifically bind to rRNA for depletion. On average, the amount of rRNA remaining in the libraries ranged from 10-30%. Several depletion methods and kits were tested to develop a more effective and robust depletion method that can be high-throughput and automated while keeping library preparation costs low and cut down on hands-on time. The KAPA RiboErase kit skipping intermediate purification and the Lucigen RNase H method resulted in an average of 1-5% remaining rRNA.