GSM8751013: TTchem_1h_dTAG; Mus musculus; OTHER
RNA was extracted by TRIzol according to manufacturer instructions. 100 μg of mouse RNA was mixed with 1 μg of 4TU-labelled yeast RNA and brought to a total volume of 100 μl with water. The mix was fragmented by adding 20 μl 1M NaOH and incubating on ice for 20 min. 80 μl of 1M Tris-HCl pH 6.8 was added to stop fragmentation and samples were cleaned up twice with Micro Bio-Spin P30 Gel Columns (BioRad, 7326250) following manufacturer instructions. Biotinylation of 4SU and 4TU residues was carried out in a total volume of 250 μl 10 mM Tris-HCl pH 7.4 and 1 mM EDTA, containing MTSEA biotin-XXlinker (Biotium, BT90066) for 30 min at room temperature in the dark. RNA was then purified by phenol:chloroform extraction, denatured for 10 min at 65°C and added to 200 μl μMACS Streptavidine MicroBeads (Miltenyi Biotec, 130-074-101). After 30 min incubation at room temperature, the mix was loaded to a μColumn in the magnetic field of a μMACS magnetic separator. Beads were washed twice in a 55C buffer containing 100 mM Tris-HCl pH7.4, 10 mM EDTA, 1M NaCl and 01% Tween20. Biotinylated RNA was eluted twice in 100 mM DTT (200 μl final volume) and cleaned up with the RNeasy MinElute kit (QIAGEN, 74204) using 1050 μl 100% ethanol and 750 μl RLT buffer to precipitate RNA <200nt. TTchem-seq libraries were prepared from 5ng of purified RNA using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® following the protocol for FFPE RNA with adapters added from the NEBNext® Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors RNA Set 1) according to manufacturer instructions. Average fragment size was determined on a TapeStation using the HS 5000 DNA kit, and concentrations determined by Qubit. Sequencing was performed single-end on a NextSeq 2000