title: GSM4802254: mRNA_Control_E14_mESC_1; Mus musculus; RNA-Seq
library_construction_protocol: To isolate mRNA, total RNAs were purified from mESCs or MEFs using the TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Briefly, mESCs of MEFs were harvested and homogenized with 1 ml of TRIzol reagent. Chloroform (200 μL/sample) was added, and the samples were mixed vigorously by hand for 15 seconds and incubated at 25 °C for 2 minutes. The mixtures were centrifuged at 12,000 rpm for 15 minutes at 4 °C, and 500 μL of each aqueous phase was transferred to a new Eppendorf tube and mixed with the same volume of isopropanol. The mixtures were incubated at 25 °C for 10 minutes to precipitate total RNAs. The samples were centrifuged at 12,000 rpm for 10 minutes at 4 °C, washed with 75% ethanol, and centrifuged again at 10,000 rpm for 5 minutes at 4°C. The RNA pellets were dried and dissolved in RNase-free water.  Finally, mRNAs were isolated from total RNA using a Magnetic mRNA isolation kit (S1550S, NEB). mRNA-seq libraries were made using NEXTflex™ Rapid Directional mRNA-Seq kit (5138-10, BIOO) from purified mRNA.