title: GSM5350991: VQFR2_total WT with Tir1/BirA o/e; 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, mouse embryonic stem cells and Drosophila melanogaster S2 cells. 4tU was used to label newly synthesized RNA from S. pombe cells. We use Drosophila S2 cells  (VQFR1-12) or S. pombe cells (VQFR13-28) 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. To be able to judge the specificity of the 4sU purification, we also added 10X of unlabelled S. cerevisiae RNA prior to the purification of Samples 1-12. Also, the total RNA, used for the purification of the 4sU-containing RNA, was fragmented prior to purification (into on average ~700 bp fragments). Total RNA-Seq libraries were generated from 1000 ng of total RNA or 14-40 ng of newly synthesized, 4sU-labelled RNA using TruSeq Stranded Total RNA LT Sample Prep Kit with Ribo-Zero Gold (Illumina, San Diego, CA), according to manufacturer's instructions. Briefly, cytoplasmic and mitochondrial ribosomal RNA (rRNA) was removed using biotinylated, target-specific oligos combined with Ribo-Zero rRNA removal beads. This step was not performed for the newly synthesized (4sU) RNA. Following purification, the depleted RNA was fragmented into small pieces using divalent cations at 94°C for 2 minutes (for newly synthesized RNA, which is already prefragmented, fragmentation was reduced to 1 minute). Cleaved RNA fragments were then copied into first strand cDNA using reverse transcriptase and random primers followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. Strand specificity was achieved by replacing dTTP with dUTP during second strand synthesis. The double stranded cDNA fragments were blunted using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK. A single 'A' nucleotide was added to the 3' ends of the blunt DNA fragments using a Klenow fragment (3' to 5'exo minus) enzyme. The cDNA fragments were ligated to double stranded adapters using T4 DNA Ligase. The ligated products were enriched by PCR amplification (30 sec at 98°C; [10 sec at 98°C, 30 sec at 60°C, 30 sec at 72°C] x 10 cycles; 5 min at 72°C). Surplus PCR primers were further removed by purification using AMPure XP beads (Beckman-Coulter, Villepinte, France) and the final cDNA libraries were checked for quality and quantified using capillary electrophoresis.