GSM4976316: PBS237; Mus musculus; RNA-Seq
Tissue was homogenized in TRIzol using a hand-held pestle homogenizer and incubated in TRIzol for at least 5 minutes. Chloroform (1:5 ratio) was added, the tubes shaken, and incubated at room temperature for 15 minutes. Samples were then centrifuged at 12,000g for 15 minutes at 4℃. The top aqueous layer was transferred to a clean tube, and the RNA was precipitated in 3M NaAc pH 5.2 (10:1 ratio), 4ul of glycogen (5mg/ml), 100% isopropanol (1:1 ratio) overnight at -80℃. The next day, the samples were centrifuged at 15,000 RPM for 20 minutes at 4℃. The resulting RNA pellet was washed once in 75% ethanol, centrifuged at 7,500g for 10 minutes at 4℃. The washed RNA pellet was dissolved in nuclease-free water. RNAseq libraries were generated using TruSeq RNA Sample Prep V2 kit (New England BioLabs) with 1ug of RNA to first obtain poly-A-enriched RNA and synthesize cDNA, and then the same library construction protocol was followed using 5ng of cDNA.