Nextseq 500 550 high output v2 kit 75 cycles cartridge

Quality of RNA extraction, yield and Library synthesis were measured using RNA ScreenTape kit (AGILENT TECHNOLOGIES), D1000 ScreenTape kit (AGILENT TECHNOLOGIES), Qubit® RNA HS Assay kit (Invitrogen), Qubit® DNA HS Assay kit (Invitrogen) were used for each specific step purpose. For mRNA library preparation, KAPA Stranded mRNA-Seq Kit with mRNA Capture Beads (kappa biosystems, KK8421, https://www.kapabiosystems.com/) was used. In brief,  1 μg of total RNA was used for the library construction, libraries were eluted in 20 μl of elution buffer, and adjusted to 10 nM. 10 μl (50%) from each sample were collected and pooled in one tube. Multiplex samples were pooled (1.5 pM including PhiX 1.5%) and loaded in a NextSeq 500/550 High Output v2 kit (75 cycles) cartridge (Illumina) and loaded on a NextSeq 500 System (Illumina), with 75 cycles and single-Read Sequencing conditions.

Lymphocyte RNA was extracted using the trizol method. For quality control of RNA extraction yield and library synthesis products, RNA Screen Tape kit (Agilent Technologies, Waldbronn, Germany), D1000 ScreenTape kit (Agilent Technologies, Waldbronn, Germany), Qubit® RNA HS Assay kit (Invitrogen, USA), Qubit® DNA HS Assay kit (Invitrogen, USA) were used for each specific step. For mRNA library preparation, KAPA Stranded mRNA-Seq Kit with mRNA Capture Beads (Kapabiosystems, KK8421, https://www.kapabiosystems.com) was used. In brief, one μg was used for the library construction; the library was eluted in 20 μl of elution buffer. Libraries were adjusted to 10 Mm; 10 μl, 50% from each sample was collected and pooled in one tube. Multiplex samples Pool (1.5 pM including PhiX 1.5%) were loaded on NextSeq 500/550 High Output v2 kit (75 cycles) cartridge (Illumina, USA) and loaded on the NextSeq 500 System (Illumina, USA), with 75 cycles and single-Read Sequencing conditions.

Lymphocyte RNA was extracted using the trizol method. For quality control of RNA extraction yield and library synthesis products, an RNA Screen Tape kit (Agilent Technologies, Waldbronn, Germany), D1000 ScreenTape kit (Agilent Technologies, Waldbronn, Germany), Qubit^® RNA HS Assay kit (Invitrogen, USA) and Qubit^® DNA HS Assay kit (Invitrogen, USA) were used for each specific step. For mRNA library preparation, poly-A selection beads followed with NEXTflex™ Rapid Directional qRNA-Seq™ Kit were used (Bio Scientific, Cambridge, UK). In brief, 1 μg was used for the library construction and the library was eluted in 20 μL elution buffer. Libraries were adjusted and pooled to a concentration of 4 nM. Multiplex sample pools including PhiX 1.5% were loaded on a NextSeq 500/550 High Output v2 kit (75 cycles) cartridge (Illumina, San Diego, CA, USA) and loaded on the NextSeq 500 System (Illumina, USA), with 75 cycles and single-read sequencing condition.