GSM4548432: PSPC1-24h-TTseq-2; Mus musculus; RNA-Seq
TT-seq was performed as described previously with some modifications (Schwalb et al., 2016; Yin et al., 2020). Cells were labeled in media for 10 minutes with 500 µM 4-thiouridine (4sU, Sigma-Aldrich, St. Louis, MO, USA.). RNA extraction was performed with TRIzol (Life Technologies, Carlsbad, CA, USA). Labelled RNAs were further biotinylated and fragmented as previously described. The fragmented RNAs were then subjected to two times of affinity purification. After first time affinity capture by M-280 dynabeads (Invitrogen), beads were washed 2 times (5-10 min each) with 0.5 ml of washing buffer (100 mM Tris pH 7.4, 10 mM EDTA, 1 M NaCl, 0.1% Tween-20) at 45 ℃, followed by another 2 times of wash with 0.5 ml of SDS washing buffer (50 mM Tri-Cl, pH8.1, 10 mM EDTA, 1% SDS) at room temperature. RNA was eluted by 50 μl SDS washing buffer at 95 °C for 5 minutes. Repeat once and combine the eluate. 50 μl pre-washed M280 beads were added to the eluate and incubated for another 20 minutes with rotation at room temperature. Beads were washed as described above. RNA was eluted with 100 µl SDS washing buffer supplemented with 0.1 M dithiotheitol (DTT) and purified as reported. Libraries were prepared by NEBNext Ultra II directional RNA library prep kits according to the standard protocols provided by the manufacturer.
GEO Accession
GSM4548432