BulkRNASeq

Bulk RNA sequencing (bulk RNA-Seq) is a high-throughput genomics method that quantifies gene expression in a mixture of cells, yielding an averaged profile across the sample. It contrasts with single-cell RNA-Seq, which resolves expression at the level of individual cells.

RNA is extracted from the tissue or culture, converted into sequencing libraries via poly(A) selection or ribosomal RNA depletion, and sequenced on platforms such as Illumina. Typical depth ranges from tens of millions of reads per sample, with paired-end reads common.

In data analysis, reads are quality-checked, aligned to a reference genome or transcriptome, and counted for each gene or transcript. Quantification can be alignment-based (STAR, HISAT2, featureCounts) or alignment-free (Kallisto, Salmon). Normalization and differential expression analysis use packages like DESeq2, edgeR, or limma.

Outputs include gene-level counts or normalized expression (TPM/FPKM), differential expression results with adjusted p-values, and various visualizations. Downstream analyses often involve pathway enrichment, gene ontology, and network or motif analyses.

Strengths of bulk RNA-Seq include robustness, established pipelines, and cost efficiency for samples with many cells. Limitations include loss of cell-to-cell heterogeneity, dependence on RNA quality, and biases from library preparation or sequencing depth. Experimental design should use replicates and proper randomization.

Applications span differential expression studies, transcriptome profiling across conditions, time courses, and biomarker discovery. Bulk RNA-Seq remains a foundational method alongside single-cell approaches for comprehensive transcriptomic analysis.