Second-generation sequencing (SGS) has become the preferred method for RNA transcriptome profiling of organisms and single cells. However, SGS analysis of transcriptome diversity (including protein-coding transcripts and regulatory non-coding RNAs) is inefficient unless the sample of interest is first depleted of nucleic acids derived from ribosomal RNA (rRNA), which typically account for up to 95% of total intracellular RNA content. Here we describe a novel microscale hydroxyapatite chromatography (HAC) normalization method to remove eukaryotic and prokaryotic high abundant rRNA species, thereby increasing sequence coverage depth and transcript diversity across non-rRNA populations. RNA-seq analysis of Escherichia coli K-12 and human intracellular total RNA showed that HAC-based normalization enriched for all non-ribosomal RNA species regardless of RNA transcript abundance or length when compared with untreated controls. Microcolumn HAC normalization generated rRNA-depleted cDNA libraries comparable to the well-established duplex specific nuclease (DSN) normalization and Ribo-Zero rRNA-depletion methods, thus establishing microscale HAC as an effective, cost saving, and non-destructive alternative normalization technique.