GSM4594572: HEK293T_RNaseHigh_rep1; Homo sapiens; RNA-Seq
For low-input RNase footprinting, cells were treated with 100 ug/ml cycloheximide for 3 min before harvesting. Cells were then rinsed twice with PBS and lysed using cold lysis buffer. The cell lysis was digested with RNase 1 (cat. no. N6901K, Lucigen) at room temperature for 1.5 hour. We added 400 ul of TRIzol (Ambion) and then 100 ul of chloroform (Sigma) to extract RNase footprints. The footprints were precipitated overnight with isopropanol.   To compare RNA fragments after digestion with different doses of RNase, we used 0.5 million HEK293T cells for the experiments. We treated the cell lysate containing 10 ug total RNA with different RNase doses (cat. no. N6901K, Lucigen): 0.5 U (low dose), 5 U (medium dose), and 500 U (high dose), respectively, at the room temperature for 1 hr. The RNA fragments were precipitated overnight with isopropanol.  For Ribosome profiling, libraries were prepared based on the published protocol in (McGlincy and Ingolia, Methods, 2017).   For RNA-seq, total RNA was isolated using the Direct-zol RNA kit (Zymo Research), and mRNAs were purified using Oligo(dT)25 magnetic beads (New England Biolabs) according to the manufacturer's instructions. Purified mRNAs were fragmented with NEBNext Mg2+ RNA fragmentation module (New England Biolabs) at 94 for 6 min. Fragmented RNAs were then precipitated overnight at -20 C by adding 0.1 volumes of 3M sodium acetate, 10 mg of glycoblue, and 1.2 volumes of isopropanol. We use the same procedures for low-input RNase footprinting and RNA-seq, precipitated RNA fragments were suspended in 5 ul of TNK reaction mixture and end-repaired at 37 C for 90 min followed by incubating at 65 C for 5 min to inactive PNK. The end-repaired fragments were 3' polyadenylated with 5 U of E. coli poly (A) polymerase (New England Biolabs) by incubating at 37 C for 2-4 hrs. The resulting polyadenylated RNA was next reverse transcribed by a modified SMAT-RT reaction with customized template-switching oligos and incubated at 42 C for 1 h, followed by 3 cycles of heating to 50 C for 2 min and then cooling to 42 C for 2 min. The enzyme was inactivated by heating at 70 C for 10 min. The RNA fragments hybridized to DNA were then removed by incubating reaction mixture at 37 C for 15 min with 5 U of RNase H (New England Biolabs). The resulting cDNA was then amplified by two rounds of PCR to generate the sequencing library. DNA library was separated and visualized in a 4% agarose gel. DNA with 15-35 bp-insert was excised and recovered using the DNA gel recover kit (Zymo Research). For Ribosome profiling, the adapter ligation were carried out based on the published protocol in (McGlincy and Ingolia, Methods, 2017). To compare RNA fragments after digestion with different doses of RNase, for the medium and high dose conditions, we selected RNA fragments with 10-40 nt to generate the sequencing library, using the same adapter ligation procedures described in ribosome profiling. For the low dose RNase condition, we extracted all RNA fragments and used A-tailing and SMARTer oligo-based template switch described above to generate the sequencing library.
GEO Accession
GSM4594572