GSM1836613: Expt6_FU1-Vs-06_DE_el__42_EDTA; Homo sapiens; RIP-Seq
One 10 cm dish of stable cell lines expressing FLAG-TOE1 variants was transfected twice with 20 nM siRNA targeting either luciferase or the 3’UTR of TOE1 using siLentFect (Bio-Rad) according to the manufacturer’s recommendations 72 h and 24 h before harvest. At ~50% confluency, cells were induced for 24 hours with tetracycline at 1 ng/ml. Cells were harvested in PBS and lysed in isotonic lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2 mM EDTA, 0.1% Triton X-100, 1 mM PMSF, 2 μg/mL aprotinin, 2 μg/mL leupeptin, 0.1 μg/ml yeast total RNA, 80 U/mL RNaseOUT (Invitrogen)) for 10 minutes on ice. Lysates were cleared at 20K x g for 15 min at 4°C. FLAG peptide was added to 1 μg/ml and lysates were incubated for 2 h with Anti-FLAG M2 beads (Sigma) at 4°C. Beads were then washed 8 times with NET2 (10 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Triton X-100). One-fifth of the beads were eluted in protein loading buffer, and four-fifths of the beads were resuspended in Trizol (Life Technologies). RNA was extracted according to manufacturer’s protocol. RNA adapters containing bar-codes and 10-11 nucleotide randomers were ligated to the 3’ ends of total and eluted RNA with T4 RNA ligase (NEB) at 16°C for 16 hours. Ligated RNA samples were treated with RQ1 RNase-free DNase (Promega) for 30 minutes at 37°C and extracted with PCA (Affymetrix). cDNA was generated from AR-17 primer with Superscript III (Life Technologies). 3’ ends were amplified by Q5 High-Fidelity DNA Polymerase (NEB) first, using snRNA gene-specific primers and AR-17, and then with primers D501 and D702 (Illumina). Libraries were purified on AMPure XP magnetic beads (Beckman), and validated by 2200 Tape Station Instrument (Agilent). Libraries were pooled at 4 nM, denatured with NaOH and diluted in HT1 buffer (Illumina) to 7 pM before sequencing with MiSeq Reagent Kit V2 Nano configuration (Illumina).
GEO Accession
GSM1836613