GSM4174237: rep2_RNA_2K; Mus musculus; RNA-Seq
The brain was rapidly removed from P28–P35 mice, rinsed in ice-cold dissection buffer (1× HBSS + 10 mM HEPES-KOH), rapidly dissected in dissection buffer ice-liquid mixture to collect cerebral cortex as described previously(Stefani et al., 2004). Both cortex hemispheres were homogenized in 900µl of homogenization buffer(Stefani et al., 2004) (10 mm HEPES-KOH, pH 7.4, 150 mm KCl, 5 mm MgCl2, 0.5 mm DTT, 100 µg/ml cycloheximide and 2 µg/ml harringtonine), containing protease and phosphatase inhibitors (cOmplete, EDTA-free Protease Inhibitor Cocktail and PhosSTOP from Roche/Sigma, cat. no. 11836170001 and 4906837001), in a pre-chilled 2-mL glass Dounce homogenizer, 20 strokes loose, 20 strokes tight, and centrifuged at low speed (2000 rcf 10 min 4°C) to pellet insoluble material(Udagawa et al., 2013). Five hundred microliters of the resulted ~700µl supernatant (cytoplasmic lysate) were used for ribosome profiling, the rest for RNA-seq. For ribosome profiling, the lysate was digested by 60 U RNase T1 (Thermo Scientific, cat. no. EN0541) and 100ng RNase A (Ambion, cat. no. AM2270) per A260 unit(Cenik et al., 2015) for 30 min at 25°C with gentle mixing. Digestion was stopped by adding 30 µl SUPERase·In (Invitrogen, cat. no. AM2694). Digested lysate was separated by sedimentation through sucrose gradients. Monosome fractions were identified, pooled, and extracted with TRIzol LS (Invitrogen, cat. no. 10296028). For RNA-seq, cytoplasmic RNA was extracted from the lysate using TRIzol LS. Ten micrograms of RNA were depleted of rRNA using Ribo-Zero Gold rRNA Removal Kit, Human/Mouse/Rat (illumina, discontinued), and fragmented by incubating with PNK buffer (NEB, cat. no. M0201S) for 10 min at 94°C. Fragmented RNA as separated on 15% urea-polyacrylamide gel, and 50-60nt fraction was collected. Ribosome profiling and RNA-seq libraries were prepared following published protocols(Heyer et al., 2015)
GEO
GSM4174237
GEO Accession
GSM4174237