GSM8321859: TTseq, INTS11-AID cells, ASO, Control 1; Homo sapiens; OTHER
Chromatin was extracted by Chromatin RNA separation and chromatin RNA was extracted by Trizol. Construction of chromatin RNA libraries. After 1h treatment with IAA, PBS was washed away for three times and replaced with fresh medium for 7h culture, and chromatin RNA was extracted to construct library 4sU-seq and TT-seq and data processing Detailed procedures of 4sU-seq and TT-seq followed previously pub- lished papers69,70. For 4sU-seq, cells were treated with 4-thiouridine (4sU) at a final concentration of 2 mM for 15 min, and total RNA was extracted by TRIzol (Invitrogen, 15596018). A mixture of 80 μg of total RNA and 2 μg of 4sU-labeled RNA from Drosophila S2 cells (as spike-in) was biotinylated in 4sU-seq biotinylation mix at room temperature for 2 h. Subsequently, RNA was purified by chloroform extraction twice and precipitated with isopropanol. After denaturation for 10 min at 65 °C, RNA was incubated with 50 μl of prewashed streptavidin mag- netic C1 beads (Invitrogen, 65002), with gentle rotation for 15 min at room temperature. Beads were washed five times with bead wash buffer (1 M NaCl, 5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 0.05% Tween 20). The 4sU-labeled RNA was eluted with 100 mM DTT and purified by a kit (Zymo Research, R1016). The RNA libraries were constructed with VAHTS Universal V8 RNA-seq Library Prep kit for MGI (Vazyme, NRM605-02). The quality of libraries was monitored by Bio-Fragment Analyzer (Bioptic, c100001) and the libraries were sequenced with MGI 2000 instrument (MGI-SEQ, BGI). For TT-seq, cells were treated with 2 mM 4sU for 7 min and quenched with direction RNA extraction by TRIzol reagent (Invitrogen, 15596018). The mixture of 80 μg total RNA and 2 μg 4sU-labeled RNA of Drosophila S2 cells (as spike-in) was fragmented using 20 μl 1 M NaOH for 23 min on ice and neutralized with 80 μl 1 M Tris-HCl (pH 6.8). RNA was biotinylated in TT-seq biotinylation mix for 30 min at room temperature. Library Construction using universal RNA-seq library prep kit (Vazyme, NRM605-02). 500ng of Chromatin RNA was used for the construction of sequencing libraries. Chromatin RNA-seq, 4sU-seq, TT-seq