Duplex specific thermostable nuclease normalization protocol

Example 2

RNA-sequencing on WM35, WM3248, M14, and SK-MEL-28 cells was performed to determine functional methylation, i.e., methylation associated with downregulated gene expression [26]. In brief, total ribonucleic acid (RNA) was isolated using the standard procedure for TRIzol® RNA extraction (Invitrogen, Bleiswijk, The Netherlands) and stored at −80° C. For total RNA sequencing, library preparation was carried out using a modified version of the Illumina “Directional mRNA-sequencing Sample Preparation” protocol with total RNA instead of mRNA. Ribosomal DNA was depleted from the DNA fraction using Illumina's Duplex-Specific Thermostable Nuclease normalization protocol for bidirectional mRNA sequencing (application note 15014673).

Example 2

RNA-sequencing on WM35, WM3248, M14, and SK-MEL-28 cells was performed to determine functional methylation, i.e., methylation associated with downregulated gene expression [26]. In brief, total ribonucleic acid (RNA) was isolated using the standard procedure for TRIzol® RNA extraction (Invitrogen, Bleiswijk, The Netherlands) and stored at −80° C. For total RNA sequencing, library preparation was carried out using a modified version of the Illumina “Directional mRNA-sequencing Sample Preparation” protocol with total RNA instead of mRNA. Ribosomal DNA was depleted from the DNA fraction using Illumina's Duplex-Specific Thermostable Nuclease normalization protocol for bidirectional mRNA sequencing (application note 15014673).