GSM5267971: Female WT Cerebral cortex replicate 1; Mus musculus; RNA-Seq
Cerebral cortex, hippocampus, hypothalamus and cerebellum were dissected, flash-frozen and kept at -80°C until use. Tissue homogenization and RNA extraction were performed using the mirVana Isolation kit (Thermo Fisher Scientific; #AM1560) according to the manufacturer's instructions. 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 closed to the 3' end gets captured for later indexed adapter ligation and PCR amplification. The processed libraries were assessed for its size distribution and concentration using BioAnalyzer High Sensitivity DNA Kit (Agilent Technologies). Pooled libraries were diluted to 2 nM in EB buffer (Qiagen) and then denatured using the Illumina protocol. The libraries were pooled and diluted to 3 nM using 10 mM Tris-HCl, pH 8.5 and then denatured using the Illumina protocol. The denatured libraries were diluted to 10 pM by pre-chilled hybridization buffer and loaded onto a TruSeq v2 Rapid flow cell on an Illumina HiSeq 2500 and run for 50 cycles using a single-read recipe according to the manufacturer's instructions.