X0M2_2_Xylem_ 0 zeb_ 2 month in soil_ rep2
After the QC procedures, mRNA from eukaryotic organisms is enriched using oligo (dT) beads. For prokaryotic samples, rRNA is removed first using a specialized kit before enrichment. The mRNA is then fragmented randomly in fragmentation buffer. First-strand cDNA was synthesised using random hexamers and reverse transcriptase. Then the second strand was generated using a custom second-strand synthesis buffer (Illumina) with dNTPs, RNase H and Escherichia coli polymerase I by nick-translation. The final cDNA library is ready after purification, terminal repair, A-tailing, adapter ligation, size selection and PCR enrichment. (Novoene protocol)