Novaseq 6000 s1 reagent kit v1

Whole transcriptome library preparation was performed using the SMARTer® Stranded Total RNA-Seq Kit v3 –Pico Input Mammalian (Takara Bio Europe, France) according to the manufacturer’s instructions. Briefly, 10 ng total RNA was fragmented, cDNA synthesis was performed using random N6 primers, multiplexing indices and adapters were added, and ribosomal cDNA depletion was performed. Due to the fragmented nature of the RNA extracted from FFPE tissue, the fragmentation step was omitted in the library preparation of RNA from FFPE tissues. AMPure XP beads (Beckman Coulter, USA) were used instead of the recommended NucleoMag NGS Clean-up and Size Select beads (Macherey-Nagel, Germany). The qualities of the cDNA libraries were assessed using the Bioanalyzer High Sensitivity DNA assay with the 2100 Bioanalyzer system (Agilent, USA). The quantities of the cDNA libraries were assessed using the 7500 Real-Time PCR System (Applied Biosystems, USA) with the KAPA Library Quantification Kits–Complete kit (ABI Prism) (Kapa Biosystems, USA). Paired-end sequencing (2 x 100 bp) was performed on a NovaSeq 6000 instrument using the NovaSeq 6000 S1 Reagent Kit v1.5 (Illumina, USA).

Total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen, USA) after grinding plant material (100–150 mg) in liquid nitrogen. The amount of RNA was determined using a Qubit fluorometer (Invitrogen, USA) using the Qubit ^™ RNA HS Assay Kit. To create barcoded RNA-Seq libraries, the NEBNext^® Ultra ^™ II RNA Library Prep Kit for Illumina^® (New England BioLabs, USA) was used according to the manufacturer’s protocol. Sequencing of the obtained libraries was performed on a high-performance sequencer NovaSeq 6000 (Illumina, USA) using the NovaSeq 6000 S1 Reagent Kit v1.5 (300 cycles).