GSM5769655
Brains were removed from RNAlater and placed in 200 ųl of 1-Thioglycerol/Homogenization Solution from the MAxwell 16 LEV Simply RNA Tissue Kit (Promega cat# AS1280). Samples were homogenized using a hand held pestle motor and plastic pestle in a microcentrifuge tube. Homogenized samples were transferred to the MAxwell 16 LEV Simply RNA Tissue Kit cartridge and processed in the Maxwell 16 Instrument (Promega AS1000) using the High Strength LEV Magnetic Rod and Plunger Bar Adaptor (Cat.# SP1070). Standard DNAse treatment was included and samples were eluted in 50 ųl H20 including RNAse inhibitors. Library construction was performed by NovoGene as follows: After the QC procedures, mRNA was enriched using oligo(dT) beads. First, the mRNA is fragmented randomly by adding fragmentation buffer, then the cDNA is synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina), dNTPs, RNase H and DNA polymerase I are added to initiate the second-strand synthesis. Second, after a series of terminal repair, A ligation and sequencing adaptor ligation, the double-stranded cDNA library is completed through size selection and PCR enrichment.