GSM8654845: KYSE-150 cells,PAK5-OE2; Homo sapiens; RNA-Seq
After the sample is qualified, eukaryotic mRNA is enriched with magnetic beads with Oligo (dT) (in the case of prokaryotes, mRNA is enriched by removing rRNA by the kit). Then mRNA was fragmented into short fragments by fragmentation buffer. A cDNA strand was synthesized using the mRNA template with six-base random hexamers, and then a two-strand cDNA was synthesized by buffer solution, dNTPs and DNA polymerase I. AMPure XP beads were then used to purify double-stranded cDNA. The purified double-stranded cDNA was then end-repaired, A tail was added and sequencing joints were connected. Then AMPure XP beads were used for fragment size selection, and finally PCR enrichment was performed to obtain the final cDNA library. After the completion of the library construction, Qubit2.0 was used for preliminary quantification, and the library was diluted to 1ng/ul. Then Agilent 2100 was used to detect the insert size of the library. After the insert size met the expectation, Q-PCR method was used to quantify the effective concentration of the library accurately (effective concentration of the library > 2nM) to ensure the quality of the library.