Bioanalyzer smear analysis tool

All sequencing was conducted at Qiagen Genomic Services, Hilden, Germany as previously described [12 (link)]. The library preparation was carried out using TruSeq^® Stranded mRNA Sample preparation kit (Cat.no. 20020594, Illumina Inc., San Diego, CA, USA). The starting material (500 ng) of total RNA was mRNA enriched using the oligodT bead system. The isolated mRNA was subsequently enzymatically fragmented. Then, first-strand synthesis and second-strand synthesis were performed, and the double-stranded cDNA was purified (AMPure XP, Cat.no. A63881, Beckman Coulter, Brea, CA, USA). The cDNA was end-repaired, 3′ adenylated, and Illumina sequencing adaptors ligated onto the fragments ends, and the library was purified (AMPure XP). The mRNA-stranded libraries were preamplified with PCR and purified (AMPure XP). The library’s size distribution was validated, and quality-inspected on a Bioanalyzer 2100 or BioAnalyzer 4200tape Station (Agilent Technologies, Santa Clara, CA, USA). High-quality libraries were pooled based on equimolar concentrations based on the Bioanalyzer Smear Analysis tool (Agilent Technologies). The library pool(s) were quantified using qPCR and optimal concentration of the library pool used to generate the clusters on the surface of a flow cell before sequencing on a NextSeq500 instrument (75 cycles) according to the manufacturer’s instructions (Illumina Inc.).

All experiments were conducted at QIAGEN Genomic Services. The library preparation was done using TruSeq^® Stranded mRNA Sample preparation kit (Illumina Inc.). The starting material (500 ng) of total RNA was mRNA enriched using the oligodT bead system. The isolated mRNA was subsequently enzymatically fragmented. Then first-strand synthesis and second-strand synthesis were performed, and the double-stranded cDNA was purified (AMPure XP, Beckman Coulter). The cDNA was end repaired, 3′ adenylated and Illumina sequencing adaptors ligated onto the fragments ends, and the library was purified (AMPure XP). The mRNA stranded libraries were pre-amplified with PCR and purified (AMPure XP). The libraries' size distribution was validated and quality inspected on a Bioanalyzer 2100 or BioAnalyzer 4200tape Station (Agilent Technologies). High-quality libraries were pooled based in equimolar concentrations based on the Bioanalyzer Smear Analysis tool (Agilent Technologies). The library pool(s) were quantified using qPCR, and the optimal concentration of the library pool was used to generate the clusters on the surface of a flow cell before sequencing on a NextSeq 500 instrument (75 cycles) according to the manufacturer instructions (Illumina Inc.).

The library was prepared using the TruSeq® Stranded mRNA Sample preparation kit (Illumina Inc) which produces a cDNA library for whole transcriptome analysis. Briefly, the starting material (500 ng) of total RNA was mRNA enriched using the oligodT bead system. The isolated mRNA was subsequently fragmented using enzymatic fragmentation. Then first strand synthesis and second strand synthesis were performed and the double stranded cDNA was purified (AMPure XP, Beckman Coulter). The cDNA was end repaired, 3’ adenylated and Illumina sequencing adaptors ligated onto the fragments ends, and the library was purified (AMPure XP). The mRNA stranded libraries were pre-amplified with PCR and purified (AMPure XP). The libraries size distribution was validated and quality inspected on a Bioanalyzer 2100 or BioAnalyzer 4200 TapeStation (Agilent Technologies). High quality libraries were pooled in equimolar concentrations based on the Bioanalyzer Smear Analysis tool (Agilent Technologies). The library pool(s) were quantified using qPCR and optimal concentration of the library pool used to generate the clusters on the surface of a flowcell before sequencing.