Table 1.

Sources of Batch Effect and Proposed Strategies to Mitigate Them

Source	Strategy to Mitigate Batch Effect
Experiment
User	Minimize users or establish interuser reproducibility in advance.
Temporal	Harvest cells or kill animals at the same time of day.
	Harvest controls and experimental conditions on the same day.
Environmental	If samples are to be collected in batches, minimize the time between batch collections.
	Use intraanimal, littermate, and cage mate controls whenever possible.
RNA isolation and library preparation
User	Minimize users or establish interuser reproducibility in advance.
Temporal	Perform RNA isolation on the same day, and avoid separate isolations over several days or weeks.
	Handle all samples in the same fashion (e.g., for freeze–thaws).
Environmental	Isolate RNA batches and prepare libraries using standard precautions to minimize contamination.
Sequencing run
Temporal	Sequence controls and experimental conditions on the same run.