title: GSM6927677: Ishikawa-NC [INC]; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA extraction in four groups (Ishikawa-NC V.S Ishikawa-HK2-siRNA, HEC1A-NC V.S HEC1A-HK2-siRNA) was used by Trizol for library preparation.1. Place the cracked sample or homogenate at room temperature for 5-10min, so that the nucleoprotein and nucleic acid are completely separated.  2. Add 0.2ml chloroform, shake vigorously for 15sec, and leave at room temperature for 3min. Centrifuge at 12000rpm for 10min at 4°C.  3. Absorb the upper water phase and transfer it to a clean centrifuge tube. Add isopropyl alcohol in the same volume and mix well  min.  4. Centrifuge at 12000rpm for 10min at 4°C and discard the supernatant.  5. Add 1ml 75% ethanol to wash the precipitation. Centrifuge at 12000rpm for 3min at 4°C and discard the supernatant. Room temperature drying  5-10min.  6. Add 30-50μl RNase-free ddH2O to fully dissolve RNA. The resulting RNA solution was placed at -70°C  Store or use immediately for follow-up tests.  7. Qubit2.0 was used to detect RNA concentration, and agarose gel was used to detect RNA integrity and genome contamination. 1. Qubit RNA detection kit was used to accurately quantify total RNA to determine the total added RNA in library construction 2. mRNA purification and fragmentation 3. Double stranded cDNA synthesis. 4. Double stranded cDNA Purification.5. End repair /dA tail addition: 6. Joint connection: 7. Purification of connection products and fragment size sorting. 8. Library amplification.