GSM5627680: m3-ipsi; Mus musculus; RNA-Seq
Olfafctory bulb tissues were microdissected from fixed and cryopreserved whole mouse brains. Approximately 5mg of the frozen tissue was incubated with proteinase K (500 μg/ml) in 500 ul of 10 mM NaCl, 500 mM Tris (pH 8.0), 20 mM EDTA, and 1% SDS at 55 ˚C for 3 hrs until the sections are completely dissolved (Korbler et al., 2003).  The acid phenol-chloroform method was applied for RNA extraction using Direct-zol™ RNA Micro prep Kit (Zymo Research). RNA quality and quantity were determined by RNA Pico BioAnalyzer (Agilent technologies). The cDNA libraries were prepared using the QuantSeq 3'mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen) as per the manufacturer's instructions. Briefly, total RNA was reverse transcribed using oligo (dT) primers. The second cDNA strand was synthesized by random priming, in a manner that DNA polymerase is efficiently stopped when reaching the next hybridized random primer, so only the fragment close to the 3' end gets captured for later indexed adapter ligation and PCR amplification. The processed libraries were assessed for their size distribution and concentration using BioAnalyzer High Sensitivity DNA Kit (Agilent Technologies). The denatured libraries were loaded onto an SP flow cell on an Illumina NovaSeq 6000 and run for 53-101 cycles using a single-read recipe according to the manufacturer's instructions.