GSM8153508: Hey-A8 cells, shSPTLC2_2; Homo sapiens; RNA-Seq
1. Freeze 2ml Eppendorf microcentrifuge tube with tissue sample and glass ball at -80°C and then grind the sample in the MM300 Mixer Mill at 30Hz for two minutes.2. Add 1 ml fresh TRIzol Reagent to the mechanically disrupted tissue sample, vortex well, and incubate the sample at room temperature for 5 minutes.3. Add 0.2 ml of chloroform to the microcentrifuge tube for homogenization. Vortex well, and incubate the sample at room temperature for 3 minutes.4. Centrifugate the sample at maximum speed on table microcentrifuge at 4°C for 5 minutes.5. Transfer the aqueous phase to a fresh 2 ml microcentrifuge tube. Add an equal volume of chloroform to the fresh microcentrifuge tube, vortex well. Spin at maximum speed on table microcentrifuge for 5 minutes.6. Transfer the aqueous phase to a fresh 2 ml microcentrifuge tube. Add an equal volume of isopropanol to the fresh microcentrifuge tube, vortex well. Spin at maximum speed on table microcentrifuge at 4°C for 10 minutes.7. Wash the pellet with 1.5 ml 70% ethanol. Spin at maximum speed on table microcentrifuge for 5 minutes.8. Dissolve the pellet in 400 μl 1xTE at 55°С well with vortex. Add an equal volume of 10 M LiCl to the solution and chill it at -20°C for several hours (or overnight).9. Spin the tube at maximum speed on table microcentrifuge at 4°C for 10 minutes. Carefully remove and discard the supernatant (contains DNA and small RNA < 200 nt).10. Wash pellet with 1.5 ml 70% ethanol, vortex well. Spin the tube at maximum speed on table microcentrifuge at 4°C for 10 minutes. Discard the ethanol, do not dry the pellet.11. Dissolve the pellet in 200-400 μl fresh RNase-free ddH2O or 1xTE. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina following the manufacturer's recommendations and index codes were added to attribute sequences to each sample.