GSM9409115: SNU638 sh-SKA2-2 rep3; Homo sapiens; RNA-Seq
Total RNA was extracted using the Trizol® reagent kit (Invitrogen, CA, USA), and the integrity (quantity and quality) and concentration were measured using NanoDropTM OneC spectrophotometer (ThermoScientific, MA, USA). Total RNA was harvested from SNU638 shCTRL and SNU638 shSKA2 stable cells. The mRNA with polyA tail was enriched by magnetic beads with OligodT. The RNA obtained was segmented by interrupting buffer, the random N6 primers were used for reverse transcription, and then the cDNA two-strand was synthesized to form double-stranded DNA. The end of the synthesized double-stranded DNA is blunted, and the 5' end is phosphorylated, while the 3' end forms a sticky end with an “A” protruding, and then a bubbling linker with a protruding “T” at the 3' end is connected. The constructed cDNA library was inspected and sequenced after being qualified. The ligated products are amplified by PCR with specific primers. The PCR product was heat-denatured into single-stranded DNA, and then a single-stranded circular DNA library was obtained by circularizing the single-stranded DNA with a bridge primer. The constructed library is qualified by a standard cDNA library and sequenced after it is qualified.