GSM5986845: dTAGSET1AB_TTseq_Rep2_2hr; Drosophila melanogaster; Mus musculus; OTHER
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 using the Direct-zol DNA/RNA Miniprep kit (Zymo Research) as per the manufacturer's protocol. gDNA was depleted using the TURBO DNA-free Kit (Thermo Fisher Scientific). An equal quantity of RNA (60-80 µg) was taken into 100 µl nuclease free water and fragmented on ice with 20 µl 1M 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. RNA libraries were prepared from 20-50 ng RNA with the Ultra II Directional RNA library prep kit, as per the manufacturer's guidelines for rRNA depleted and FFPE RNA (NEB).