GSM4594602: Monocyte_RFP_rep2; Homo sapiens; RNA-Seq
For 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. 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 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).
GEO Accession
GSM4594602