title: GSM9057743: HCC-LM3, 45℃+G1_3; Homo sapiens; RNA-Seq
library_name: GSM9057743
library_construction_protocol: Lysate the cells with 1mL TRizol, transfer to a 1.5 mL RNase-free EP tube, invert 10-15 times, and place on ice for 5 minutes. Centrifuge at 12000×g, 4°C, for 5 minutes (place another set of tubes during centrifugation). After centrifugation, transfer the supernatant (1000μl) to a new tube. Add 200 μL chloroform to each tube in a TRizol:chloroform ratio of 5:1 (avoid light), invert 10-15 times, incubate on ice for 5 minutes, centrifuge at 4°C, 10,000 ×g for 15 minutes. Transfer the supernatant (400 ~ 500 μL) to a new EP tube. Add an equal volume of isopropanol, invert a few times, incubate on ice for 10 minutes, centrifuge at 4°C, 10,000 ×g for 10 minutes, visible white flocculent precipitate. Discard the supernatant, add 1 mL of 75% ethanol, resuspend the precipitate, invert evenly, centrifuge at 4°C, 10,000 ×g for 5 minutes, remove the supernatant, air-dry on a clean bench for 10 minutes, add RNA-free H2O (add 30μl for minimal precipitate, 100-300μl for abundant precipitate), gently resuspend. Use a reverse transcription kit, reverse transcribe 1ug RNA for subsequent experiments, and store the reverse transcribed product at -20°C. RNA libraries for RNA-seq were prepared using SMARTER mRNA-Seq Library Prep Kit following the manufacturer's protocols.