Nebnext ultra 2 directional rna library prep kit for with unique dual index primer pairs

The quality check, sequencing and provision of data as FASTQ files were carried out by StarSEQ GmbH (Mainz, Germany) as described in our previous work [54 (link)]. An Agilent RNA 6000 Nano Kit (Cat. No. 5067-1511) and Agilent 2100 Bioanalyzer (kit and device from Agilent, Santa Clara, CA, USA) were used for measuring the RNA integrity number (RIN). By using a Qubit RNA High Sensitivity (HS) Assay Kit (Cat. No. Q32855) and Qubit 4 Fluorometer (kit and device from Thermo Fisher Scientific Inc., Waltham, MA, USA), the concentration of RNA was determined. For library preparation, an NEBNext Ultra II Directional RNA Library Prep Kit for Illumina with unique dual index primer pairs (Cat. No. E7760L, New England BioLabs Inc., Ipswich, MA, USA) was used, and the concentration as well as quality were defined with a Qubit 4 Fluorometer by using a Qubit dsDNA HS Assay Kit (Cat. No. Q32854, Thermo Fisher Scientific Inc., MA, USA) and QIAxcel Instrument with QIAxcel ScreenGel 1.5.0 Cartridge (Qiagen N.V., Venlo, Netherlands). The paired-end sequencing of RNA (150 bp read length, approximately 25 million reads per sample (usually more)) was run on Illumina NextSeq 2000 (Illumina, San Diego, CA, USA).

Steps described in this section were completely performed by StarSEQ GmbH (Mainz, Germany). Measurement of RNA integrity number (RIN) was performed with Agilent RNA 6000 Nano Kit (Cat. No. 5067–1511) and Agilent 2100 Bioanalyzer (kit and device from Agilent, California, USA). RNA concentration was defined by using Qubit RNA High Sensitivity (HS) Assay Kit (Cat. No. Q32855) and Qubit 4 Fluorometer (kit and device from Thermo Fisher Scientific Inc., Massachusetts, USA). Library preparation was done with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina with unique dual index primer pairs (Cat. No. E7760L, New England BioLabs Inc., Massachusetts, USA). Concentration and quality of library preparation was measured with Qubit 4 Fluorometer by using Qubit dsDNA HS Assay Kit (Cat. No. Q32854, Thermo Fisher Scientific Inc., Massachusetts, USA) and QIAxcel Instrument with QIAxcel ScreenGel 1.5.0 Cartridge (Qiagen N.V., Venlo, Netherlands). RNA sequencing (paired-end, 150 bp read length, approximately 25 million reads per sample (usually more)) was performed with Illumina NextSeq 2000 (Illumina, California, USA). Data was provided as FASTQ files.