title: Illumina HiSeq 4000 paired end sequencing; Examination of mRNA decay rate in mouse embryonic fibroblasts
design: Examination of mRNA decay rate in mouse embryonic fibroblasts
library_construction_protocol: MEFs were isolated from mouse embryos at E14.5 and were cultured at 37° C in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Total RNAs were extracted with Isogen II according to the manufacturer's protocol (Nippon Gene). To measure mRNA stability, cells were treated with Actinomycin D (2.5 ug/mL) and total RNA was extracted at 0, 4 and 8h after the treatment. 100 ng of total RNA were used for RNA-seq library preparation with a TruSeq Stranded mRNA Library Prep Kit for NeoPrep (NP-202-1001, Illumina), which allows polyA-oligo(dT)-based purification of mRNA, according to the manufacturer's protocol, with minor modification and optimization as follows. Custom dual index adaptors were ligated at the 5' and 3'-ends of the library, and PCR was performed for 11 cycles.