title: GSM8377890: E14-T3G-TND-dTAG-rep4; Drosophila melanogaster; Mus musculus; OTHER
library_construction_protocol: cTT-seq was performed largely as described previously (Gregersen et al., 2020). In brief, 9 million ESCs and 3 million Drosophila SG4 cells were labelled with 500 µM 4-thiouridine (4sU, Glentham Life Sciences) for 15 min and harvested into TRIzol reagent. 4sU-labelled mouse and Drosophila cells were mixed and RNA was extracted and depleted from any gDNA carry-over using the Direct-zol RNA Miniprep Plus kit (R2072 Zymo Research) as per the manufacturer's protocol. An equal quantity of RNA (80-100 µg) was taken into 100 µl nuclease free water and fragmented on ice with 20 µl 1 M NaOH for 20 min. Fragmentation was stopped with 80 μl 1 M Tris, pH 6.8 and the RNA was cleaned up with Micro Bio-Spin P-30 gel columns (Biorad). RNA was biotin-labelled with 50 μl 0.1 mg/ml MTSEA biotin-XX linker (Biotium) with 3 μl biotin buffer (833 mM Tris HCl, pH 7.4, 83.3 mM EDTA) for 30 min at RT. Biotin-labelled RNA was purified with a 1:1 ratio of phenol/chloroform/isoamyl alcohol (Thermo Fisher Scientific). Streptavidin pull-down was performed with the μMACS Streptavidin Kit (Miltenyi Biotec), washing the columns three times with 55 °C pull-down wash buffer (100 mM Tris HCl, pH 7.4, 10 mM EDTA, 1 M NaCl and 0.1% Tween 20) and 3x RT pull down wash buffer. Biotin-labelled RNA was eluted with 100 μl elution buffer (100 mM DTT in nuclease-free water) and cleaned up with the RNeasy MinElute Cleanup kit (QIAGEN), adjusting the amount of ethanol to capture RNA < 200 nucleotides in length. RNA was quantified using the Qubit RNA HS assay kit and RNA libraries were prepared from 20 ng RNA with the Ultra II Directional RNA library prep kit, as per the manufacturer's guidelines for rRNA depleted and FFPE RNA (NEB).