GSM7663980: C57BL/6, morphine, rep 1; Mus musculus; RNA-Seq
Total RNA was extracted using Trizol reagent (Invitrogen,15596018) The cell lysis or the extracted total RNA was mixed with 1 μl of 10 μM Oligo-dT primer and 1 μl of 10 μM dNTP reagent and then incubated at 72℃ for 3 min. The reverse transcription reaction was performed in 1X Super Script II first-strand buffer system with 200 U of Super Script II reverse transcriptase, 0.1 μl of TSO (100 μM), 20 U RNAse Inhibitor, 0.75ul of DTT (100 mM), 3 μl of Betaine (5 M) and 0.09 μl of MgCl2 (1 M). The full length cDNA was synthesized at 42℃ for 90 min and then with 10 cycles of 50℃ for 2 min and 42℃ for 2 min. Finally, the product was incubated at 70℃ for 15 min to inactive the enzyme activation. After amplified the synthesized cDNA with 10 to 15 cycles of PCR amplification, the product was purified and used for library construction. After analyzed by Agilent Bioanalyzer and quantified by real time PCR, the library was sequenced with Illumina Novaseq platform.