title: GSM6604292: WT Input 72020; Mus musculus; RNA-Seq
library_construction_protocol: Mouse embryonic neocortices at postnatal day 0 (P0) were dissected in ice-cold HBSS media supplemented with 5mg/ml glucose and 20 mM HEPES (pH 7.2), followed by freezing on dry ice. Three mouse cortices were pooled and homogenized in freshly prepared polysome extraction buffer supplemented with EDTA-free protease inhibitor, RNaseOut, DTT, and cycloheximide. Lysis was performed by pipetting gently up and down on ice for 15 minutes.  Lysate was centrifuged at 5k rpm for 10 min at 4C and the resulting supernatant was spun at 14k for 5 min at 4C.  Following quantification, 120 ug neocortical input was layered onto 10-50% sucrose density gradients in thin wall polypropylene tubes. A portion (25%) was stored for processing as input. Gradients were spun at 39K rpm at 4C for 120 min.  Polysome profiles were generated with a continuous flow Fluorinert FC-40 and A254 recording with a Brandel fraction collector with UV absorbance recorder.  Samples were frozen at -80C until use.  Pooled monosomes (40S-60S-80S) and polysomes fractions were pooled for isolation of RNA by extraction with TRIzol LS.  RNA was precipitated with one volume of isopropanol/glycogen, then washed with 75% ethanol and air-dried.  Following resuspension, genomic DNA was destroyed with DNase treatment. Illumina TruSeq RNA Library Kit