GSM8566002: PNUTSdTAG_5minTTseq_UNT_rep3; Drosophila melanogaster; Mus musculus; OTHER
cTT-seq was performed largely as described previously (Gregersen et al., 2020). In brief, 1x10^8 ESCs and 30x10^6 Drosophila SG4 cells were labelled with 500 µM 4-thiouridine (4sU, Glentham Life Sciences) for 5 min and harvested into TRIzol reagent. 4sU-labelled mouse and Drosophila cells were mixed and RNA was isolated by phenol:chloroform exctraction using MaXtract High Density tubes (Quagene). The purified RNA was depleted from any gDNA carry-over using the Turbo DNase-free kit as per the manufacturer's protocol. 300 µg of RNA was taken into 300 µl nuclease free water and fragmented on ice with 60 µl 1 M NaOH for 20 min. Fragmentation was stopped with 240 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 150 μl 0.1 mg/ml MTSEA biotin-XX linker (Biotium) with 9 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).