GSM4232977: Brain, C2; Rattus norvegicus; RNA-Seq
We combined the use of TRIzol and silica-based columns. Tissue micropunches were sonicated in 250uL of TRIzol™ Reagent (Thermofisher Scientific, Waltham, MA). 50uL of chloroform was added to homogenate and incubated at room temperature for 5 minutes to facilitate phase separation. Samples were centrifuged at 12,000 x g for 15 minutes at 10°C. The aqueous layer (~100uL) was pipetted into a fresh microcentrifuge tube and mixed with an equal volume of ice-cold 70% ethanol to precipitate mRNA. The mixture was then pipetted onto a silica-based column membrane (Invitrogen™ PureLink™ RNA Mini Kit, Thermofisher Scientific, Waltham, MA). Samples were centrifuged at 12,000 x g for 30 seconds at 10°C and the supernatant was discarded. Samples were washed as directed by kit instructions. RNA was eluted into a fresh collection tube with 30uL of nuclease-free water. RNA concentration and quality were assessed using a spectrophotometer. All samples had 80-110 ng/uL, a 260/280 ratio of 2.0-2.1, and a 260/230 ratio of 1.8-2.0. Samples were stored in a freezer at -80°C until processing. Novogene Corporation (Sacramento, CA) prepared our libraries. RNA libraries were prepared for sequencing using standard Illumina protocols RNA-Seq, 250-300 bp insert cDNA library, Illumina platform, paired-end 150 bp
GEO Accession
GSM4232977