Disclosed herein include systems, methods, compositions, and kits for whole transcriptome analysis (WTA) with random priming and extension (RPE). The RPE-based WTA method can comprise hybridizing random primers with a plurality of first strand barcoded polynucleotides associated with a solid support and extending the random primers to generate a plurality of extension products. The method can comprise amplifying the plurality of extension products to generate a sequencing library.