title: GSM4830167: TT-SLAM-Seq_ESC_6h_dTAG13_rep1; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was extracted with Trizol and 24:1 chloroform-isoamylalcohol, precipitated with 2-propanol with 0.1 mM DTT (final concentration) and 1 µg glycogen. RNA was washed with 75 % EtOH with 0.1 mM DTT and eluted to H2O with 1 mM DTT. For enrichment of nascent transcripts with TT-SLAM-Seq, 50 µg of total RNA was used for each sample. RNA was fragmented with Magnesium RNA Fragmentation Module (NEB), precipitated with Na-acetate, glycogen and ethanol in presence of 0.1 mM DTT in -80C, washed with 75 % ethanol (0.1 mM DTT), and resuspended in water. RNA was biotinylated in 200 mM HEPES pH 7.5 and 10 mM EDTA with 5 µg of MTS-biotin (Biotium) by incubating in room temperature and protecting from light. Unbound biotin was removed from samples with phenol:chloroform extraction and precipitated from aqueous phase with NaCl and glycogen. Biotinylated RNA was denatured in 65C for 10 minutes and captured with µMACS streptavidin beads by incubating 15 minutes in rotation while keeping samples protected from light. µMACS columns were equilibrated with nucleic acid equilibration buffer and two times with biotinylation buffer (20 mM HEPES, 1 mM EDTA, pH 8). Beads were transferred to columns, washed three times with wash buffer (100 mM Tris-HCl pH7.5, 10 mM EDTA, 1 M NaCl and 0.1 % Tween 20) and labeled RNA was eluted with 100 mM DTT. RNA was cleaned up with RNEasy Minelute colums (Qiagen) and eluted to RNAse-free water with 1 mM DTT. RNA was alkylated with buffer containing 10 mM iodoacetamide, 50 mM NaPO4 (pH 8) and 50 % DMSO and by incubating reaction in 50 C for 15 minutes followed by quenching with 20 mM DTT. RNA was precipitated with glycogen, sodium acetate and ethanol, treated with Turbo DNAse (Invitrogen), and prepared into sequencing libraries with NEBNext Ultra II Directional RNA Library Prep Kit and NEBNext Multiplex Oligos (NEB), according to manufacturer's instructions, except using 8 minutes incubation time in fragmentation step. NEBNext Ultra II Directional RNA Library Prep Kit