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Table 1 Steps in the RNA-Seq workflow and potential contributions to experimental variance. At each step in an RNA-Seq experiment, different parameters can introduce unwanted variance that obscure the results of gene expression analyses. Detailed discussions of these parameters are given by the sources in the References column

From: Expression analysis of RNA sequencing data from human neural and glial cell lines depends on technical replication and normalization methods

Step in RNA-Seq Workflow Potential Contributions to Variance Reference(s)
1. Experimental Design number of replicate samples; genetic background of samples [9]
2. RNA Isolation RNA integrity number (RIN) value (RNA quality); isolation method [1, 34]
3. Library Preparation initial quantity of RNA template; RNA processing: polyA+ (mRNA enrichment), rRNA (rRNA depletion); preliminary amplification steps [1, 34]
4. Sequencing sequencing platform; depth of coverage; software for base-calls [42]
5. Preprocessing trimming adapter sequences and/ or low quality reads [1]
6. Mapping quality of reference genome, stringency [1, 34]
7. Normalization method [8, 35, 37]
8. Statistical Analysis method; stringency [1, 7]