title: GSM5350962: U2OS_Fl-SUPT3H_1; Homo sapiens; 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 U2OS cells, 4sU RNA-Seq libraries were generated from 15ng of purified, newly synthesized RNA using Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus kit and IDT for Illumina RNA UD Indexes, Ligation (Illumina, San Diego, USA, Cat# 20040525 and 20040553/4, respectively), according to manufacturer's instructions. Briefly, abundant ribosomal RNAs were depleted by hybridization to specific DNA probes and enzymatic digestion. The depleted RNAs were purified and fragmented using divalent cations at 94°C for 2 minutes. After random hexamers annealing, fragmented RNAs were then reverse transcribed into first strand complementary DNA (cDNA). Second strand cDNA synthesis further generated blunt-ended double-stranded cDNA and incorporated dTTP in place of dUTP to achieve strand specificity by quenching the second strand during amplification. Following A-tailing of DNA fragments and ligation of pre-index anchors, PCR amplification was used to add indexes and primer sequences and to enrich DNA libraries (30 sec at 98°C; [10 sec at 98°C, 30 sec at 60°C, 30 sec at 72°C] x 12 cycles; 5 min at 72°C). Surplus PCR primers were further removed by purification using SPRIselect beads (Beckman-Coulter, Villepinte, France, Cat# B23319) and the final libraries were checked for quality and quantified using capillary electrophoresis.