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pCHMA20-rep2_TRANSCRIPTOMIC

Illumina HiSeq 2500

RNA-Seq

TRANSCRIPTOMIC

PolyA

single

Total RNAs were purified from mESCs using the TRIzol reagent Invitrogen according to the manufactureru0026's protocol. Briefly, mESCs cultured in 6-well plates were harvested and homogenized with 1 ml of TRIzol reagent. Chloroform 200 microliter/sample was added, and the samples were mixed vigorously by hand for 15 sec and incubated at 25degree Celsius for 2 min. The mixtures were centrifuged at 12,000 rpm for 15 min at 4degree Celsius, and 500 microliter of each aqueous phase was transferred to a new Eppendorf tube and mixed with the same volume of isopropanol. The mixtures were incubated at 25degree Celsius for 10 min to precipitate total RNAs. The samples were centrifuged at 12,000 rpm for 10 min at 4degree Celsius, washed with 75% ethanol, and centrifuged again at 10,000 rpm for 5 min at 4degree Celsius. The RNA pellets were dried and dissolved in RNase-free water. For mRNA-seq library preparation, mRNAs were isolated from total RNA using a Magnetic mRNA isolation kit E7490, NEB, and libraries were prepared using a NEXTflex apid directional RNA-seq kit E7760, NEB. The libraries were sequenced using an Illumina HiSeq 2500 platform. The libraries were generated from two sets of biological replicates.