GSM4437400: left hippocampus, wildtype 3; Mus musculus; RNA-Seq
We dissected the left hippocampi of 46-week-old, male, wild-type littermates. We immediately flash froze the tissue in liquid nitrogen and stored it temporarily at -80°C. We later homogenized the tissue in TRIzol Reagent (ThermoFisher Scientific) with the Polytron PT 10-35 GT (Kinematica). Lastly,we isolated the RNA by chloroform phase separation, precipitated it with 2-propanol, and washed it with 75% ethanol. We eluted the purified RNA in water. We reverse transcribed 1 ug of total RNA with the partial P7 adapter (Illumina_4N_21T) and dNTPs with the addition of spiked-in azido-nucleotides (AzVTPs) at 5:1. We click-ligated the p5 adapter (IDT) to the 5′ end of the cDNA with CuAAC. We then amplified the cDNA for 21 cycles with Universal primer and 3′ indexing primer and purified it on a 2% agarose gel by extracting amplicon from 200-300 base pairs. We pooled the libraries and sequenced single-end, 75 base-pair reads on a Nextseq 550 (Illumina).
GEO Accession
GSM4437400