A method for tagging a cDNA polynucleotide, the method comprising: - providing a double-stranded mRNA:cDNA hybrid comprising a first strand cDNA polynucleotide having a 5' end and a 3' end and hybridized to a complementary mRNA having a 5' end and a 3' end, wherein the 5' end of the first strand cDNA polynucleotide comprises a first adapter sequence or complement thereof; - synthesizing one or more second strand cDNA polynucleotides that are complementary to and hybridized to the first strand cDNA polynucleotide, wherein synthesizing comprises: (i) contacting the mRNA:cDNA hybrid with an enzyme comprising RNase H activity, thereby producing mRNA fragments hybridized to the first strand cDNA, and (ii) contacting the mRNA fragments with a DNA polymerase, thereby extending the mRNA fragments in a template-directed polymerase reaction, wherein the template is the first strand cDNA polynucleotide and forming a double-stranded cDNA polynucleotide; - optionally contacting the one or more second strand cDNA polynucleotides with a DNA ligase; and - contacting the double-stranded cDNA polynucleotide with an adapter-loaded tagmentase, thereby forming a reaction mixture comprising a tagged double-stranded cDNA polynucleotide comprising a first end and a second end, wherein the first end comprises the first adapter sequence and complement thereof, and the second end comprises a second adapter sequence and complement thereof, wherein the contacting the double-stranded cDNA polynucleotide with the adapter-loaded tagmentase is performed in a reaction mixture that contains a homo adapter-loaded tagmentase comprising two identical polynucleotide adapters and does not contain adapter-loaded tagmentases having a different adapter. - providing a double-stranded mRNA:cDNA hybrid comprising a first strand cDNA polynucleotide having a 5' end and a 3' end and hybridized to a complementary mRNA having a 5' end and a 3' end, wherein the 5' end of the first strand cDNA polynucleotide comprises a first adapter sequence or complement thereof; - synthesizing one or more second strand cDNA polynucleotides that are complementary to and hybridized to the first strand cDNA polynucleotide, wherein synthesizing comprises: (i) contacting the mRNA:cDNA hybrid with an enzyme comprising RNase H activity, thereby producing mRNA fragments hybridized to the first strand cDNA, and (ii) contacting the mRNA fragments with a DNA polymerase, thereby extending the mRNA fragments in a template-directed polymerase reaction, wherein the template is the first strand cDNA polynucleotide and forming a double-stranded cDNA polynucleotide; (i) contacting the mRNA:cDNA hybrid with an enzyme comprising RNase H activity, thereby producing mRNA fragments hybridized to the first strand cDNA, and (ii) contacting the mRNA fragments with a DNA polymerase, thereby extending the mRNA fragments in a template-directed polymerase reaction, wherein the template is the first strand cDNA polynucleotide and forming a double-stranded cDNA polynucleotide; - optionally contacting the one or more second strand cDNA polynucleotides with a DNA ligase; and - contacting the double-stranded cDNA polynucleotide with an adapter-loaded tagmentase, thereby forming a reaction mixture comprising a tagged double-stranded cDNA polynucleotide comprising a first end and a second end, wherein the first end comprises the first adapter sequence and complement thereof, and the second end comprises a second adapter sequence and complement thereof, wherein the contacting the double-stranded cDNA polynucleotide with the adapter-loaded tagmentase is performed in a reaction mixture that contains a homo adapter-loaded tagmentase comprising two identical polynucleotide adapters and does not contain adapter-loaded tagmentases having a different adapter. <EOS>