GSM9060257: Primary astrocytes, Control, 24H,rep 2; Mus musculus; RNA-Seq
Total RNA was extracted using Trizol reagent (Invitrogen,15596018) The cell lysis or the extracted total RNA was mixed with 1ul of 10uM Oligo-dT primer and 1ul of 10uM dNTP reagent and then incubated at 72℃ for 3min. The reverse transcription reaction was performed in 1X Super Script II first-strand buffer system with 200U of Super Script II reverse transcriptase，0.1ul  of TSO （100uM），20U RNAse Inhibitor，0.75ul of DTT（100mM），3ul of Betaine（5M）and 0.09ul of MgCl2（1M）. The full length cDNA was synthesized at 42℃ for 90min and then with 10 cycles of 50℃ for 2min and 42℃ for 2min. Finally, the product was incubated at 70℃ for 15min to inactive the enzyme activation. After amplified the synthesized cDNA with 10 to15 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.