GSM8989181: Flag empty vector Mock Input sample, Replicate 1; Homo sapiens; RNA-Seq
1.Add 1 ml Trizol into each sample (Total 8 samples, including IP and Input samples) 2. Add 0.2 ml chloroform into each sample. Shake the tubes between two racks vigorously. 3. Centrifuge at highest speed for 28 min at 4 °C. 4. Pipetting 0.35 ml from upper aqueous phase and add into new 1.5-ml tube. Add the same volume of isopropanol (2-propanol). Shake gently. 5. Centrifuge at highest speed for 10 min at 4 °C. 6. Dump the liquid. Add 1 ml 70% ETOH. 7. Centrifuge at highest speed for 2 min at 4 °C. 8. Dump the liquid. Centrifuge again at highest speed for 1 min at 4 °C. 9. Vacuum the rest liquid. Be careful and do not suck up RNA samples. Rest the tube at RT without lid for 10 min. 10. Add 20 ul sterile H2O to resolve RNA sample. 11. Measure the concentration. Purified RNA were subjected to RNA quality control test using Qubit Flex Fluorometer and Agilent 2100 Bioanalyzer. 100 ng total RNA was used for RNA-Seq Library preparation with QIAseq® FastSelect™ RNA Library Kit. Briefly, ribosomal RNA (rRNA) depletion was carried out using the QIAseq FastSelect rRNA HMR Kit (334387, Qiagen), following the manufacturer's instructions. cDNA synthesis was performed using the QIAseq Low Input RNA Library Kit (334205, Qiagen), employing both the N6-T RT Primer (random hexamer) and the ODT-T RT Primer (oligo-dT primer). Library amplification/indexing were carried out with QIAseq UX Index kit UDI-A (331815, Qiagen), which incorporates unique 10-base dual indices (UDIs) into each library. The final library quality control test was again conducted using the Qubit Flex Fluorometer and the Agilent 2100 Bioanalyzer.