GSM5764210: R21105190LR01-KYSE450-NC-2; Homo sapiens; RNA-Seq
TRIzol extraction RNA libraries were prepared for sequencing using standard Illumina protocols.RNA purification, reverse transcription, library construction and sequencing were performed in Mingma Technologies Co., Ltd at Shanghai according to the manufacturer's instructions (Illumina). The mRNA-focused sequencing libraries from total RNA were prepared using VAHTS mRNA-seq v3 Library Prep Kit (Vazyme). PolyA mRNA was purified from total RNA using oligo-dT-attached magnetic beads and then fragmented by fragmentation buffer. Taking these short fragments as templates, first strand cDNA was synthesized using reverse transcriptase and random primers, followed by second strand cDNA synthesis. Then the synthesized cDNA was subjected to end-repair, phosphorylation and 'A' base addition according to Illumina's library construction protocol. Then Illumina sequencing adapters were added to both size of the cDNA fragments. After PCR amplification for DNA enrichment, the AMPure XP Beads (Beckmen) were used to clean up the target fragments of 200–300 bp. After library construction, Qubit 2.0 fluorometer dsDNA HS Assay (Thermo Fisher Scientific) was used to quantify concentration of the resulting sequencing libraries, while the size distribution was analyzed using Agilent BioAnalyzer (Agilent). Sequencing was performed using an Illumina Novaseq 6000 following Illumina-provided protocols for 2x150 paired-end sequencing in Mingma Technologies Co., Ltd at Shanghai.