RRNA depletion with Streptomyces coelicolor A3(2) specific probes

Organisms

This protocol has been tested with Streptomyces coelicolor A3(2) MT1110

Description

rRNA depletion using commercial kits targeting generic bacterial sequences is not 100% efficient in the High GC organism S. coelicolor.

Depletion using the NEBNext RNA Depletion Core Reagent Set with RNA Sample Purification Beads (https://www.neb.com/en-gb/products/e7870-nebnext-rna-depletion-core-reagent-set-with-rna-sample-purification-beads#Product%20Information) and custom DNA probes designed for S. coelicolor is more effective than the QIAseq FastSelect 5S/16S/23S kit (Qiagen #335927); NEBNext rRNA Depletion Kit (Bacteria) (NEB #E7850); Illumina Ribo-zero rRNA Removal Kit (Bacteria) (Illumina #MRZMB12424, discontinued). The Illumina kit tested was subsequently replaced by the Illumina Ribo-Zero Plus rRNA Depletion Kit (Illumina #20040526). Additionally three abundant non-mRNA RNAs can be depleted (Table 1).

Table 1. Abundant non-mRNA RNAs in S. coelicolor.

Materials

Single stranded DNA probes (Table 2) at 2µM concentration in IDTE pH 7.5 (10mM Tris-HCl / 0.1mM EDTA). Integrated DNA Technologies can produce these from their 25nmole DNA oligo pool product. Probes are listed in Table 2 in the Notes section below.

NEBNext RNA Depletion Core Reagent Set with RNA Sample Purification Beads (https://www.neb.com/en-gb/products/e7870-nebnext-rna-depletion-core-reagent-set-with-rna-sample-purification-beads#Product%20Information).

Protocols

As per manufacturer’s instructions.

Notes

The following histograms illustrate effectiveness of rRNA depletion using various methods. Histograms show percentage of bases aligned to NCBI reference sequence NC_003888.3 using Hisat2 program classified as follows - Coding = RNA aligned to coding sequences; Transcribed not translated = RNA aligned to rRNA, tRNA and sRNA; Intergenic = RNA aligned to 5' or 3' UTRs or between annotated sequences.

QIAseq FastSelect 5S/16S/23S kit

NEBNext rRNA Depletion Kit (Bacteria)

Illumina Ribo-zero rRNA Removal Kit (Bacteria)

rRNA depletion with S. coelicolor specific probes