title: GSM5350964: WT_mESC_1; Mus musculus; RNA-Seq
library_construction_protocol: We purify newly transcribed RNA by adding 4sU (4-thiouridine), a metabolic analogue of uridine, to the cell culture medium for 15 minutes before collecting the cells. The cells readily take up 4sU and incorporate it into RNA transcribed during the time period of 15 minutes. This protocol is used to label newly transcribed RNA in both, human U2OS cells and mouse embryonic stem cells. 4tU was used to label newly synthesized RNA from S. pombe cells which served as a external spike-in for normalization. The incorporation of 4sU (with its specific thiol group) into newly transcribed RNA allows to separate the 4sU-containing RNA from the total RNA using biotinylation and Streptavidin-coated beads. The total RNA, used for the purification of the 4sU-containing RNA, was fragmented prior to purification (into on average ~700 bp fragments). For mESC, 4sU RNA-seq libraries were generated from 15 ng of purified, newly synthesized RNA using TruSeq Stranded Total RNA LT Sample Prep Kit with Ribo-Zero Gold (Illumina, San Diego, CA, Cat# RS-122-2301) according to the Illumina protocol with the following modifications. 4sU-labelled RNA was cleaned up using 1.8X RNAClean XP beads and fragmented using divalent cations at 94°C for 1 minutes without depletion of rRNA. While, double stranded cDNA synthesis and adapter ligation were performed according to manufacturer instructions, the number of PCR cycles for library amplification was reduced to 10 cycles. After purification using SPRIselect beads (Beckman-Coulter, Villepinte, France, Cat# B23319), the libraries were sequenced with 1x 50 base pairs on a HiSeq4000 machine (Illumina).