GSM3172949: FM2; Mus musculus; RNA-Seq
Total RNA was extracted from primary microglia with Trizol reagent (Invitrogen) and contaminating DNA was depleted with clearance procedure according to the manufacturer's protocol with Riboclear™  plus (GeneAll). Full length cDNA was generated following manufacturers recommended protocols of the SMARTer-Seq v4 Ultra Low input RNA Kit for sequencing (Clontech, 634888). First-strand cDNA synthesis was initiated by adding 1 μl of 3' SMART CDS primer II A at 72℃ for 3 min from 10 ng of total RNA. The second strands were synthesis by adding SMARTer-seq v4 oligo and SMARTScribe reverse transcriptase, and the reaction was incubated at 42℃ for 90 min, followed by inactivation at 70℃ for 10 min. Double-strand cDNA was amplified by PCR for 8 cycles and purified using Agencourt AMPure beads(Beckman, A63881).   mRNA-seq library is was generated following manufacturers recommended protocols of the Nextera XT DNA library preparation kit (illumina, FC-131-1024). The cDNA was subjected to tagmentation (simultaneous fragmentation and tagging with sequencing adapters) and amplified by PCR using Index Primers from the Nextera XT DNA Index Kit (Illumina, FC-131-1001). After PCR, the DNA library was purified using AMPure beads and the quality was assessed using an Agilent 2100 Bioanalyzer.  The DNA libraries from individual samples were quantified using KAPA Library Quantification kit (KAPA biosystems, KK4854) followed by pooling. All libraries were sequenced on an illumina Hiseq2500 instrument to generate dual indexed 100 bp paired reads, generating an average 58 million reads for each sample.
GEO Accession
GSM3172949