GSM4332032: light-poly; Homo sapiens; RNA-Seq
[RNA prep] HEK293T cells seeded at 3.0 × 10^6 cells grown in three 10cm dishes were treated with 100 ug/mL cycloheximide (CHX) for 10 min at 37 °C. Cells were washed once with ice-cold PBS containing 100 ug/mL CHX and then resuspended in 450 uL of polysome lysis buffer (20 mM HEPES-KOH (pH 7.5), 150 mM KCl, 5 mM MgCl2, 0.5 mM EGTA, 0.5 % NP-40, 1×Protease inhibitor cocktail) containing 100 g/mL CHX. After 10 min of incubation on ice, the lysates were mixed with 50 uL of 1:1 mixture of 10 % Triton X-100/ 10 % Na-deoxycholate and then centrifuged at 204,000 × g for 10 min at 4 °C. The RNA concentration was quantified using QuantiFluor RNA System (Promega), and diluted supernatant containing the same amount of RNA was loaded onto 10-50 % sucrose gradients (20mM HEPES-KOH (pH 7.5), 150 mM KCl, 5 mM MgCl2, 1 mM EGTA, 100 ug/mL CHX) and separated into fractions by centrifugation at 37,000 rpm for 90 min using an SW41Ti rotor. Ten percent of the diluted supernatant was used as an input. Fractions were collected using piston gradient fractionator (Biocomp) while measuring A254. Collected fractions were mixed with 1 volume of Solution D and isopropanol precipitated in the presence of glycogen.   [Library prep]GST-eIF4Ek119A (100 μg) was immobilized on 50 μL of MagneGST Glutathione Particles (Promega) at 10 °C for 2 hr in 200 μL of 4E pull-down (4E-PD) buffer (10 mM Tris-HCl (pH 8.0), 100 mM KCl, 2 mM EDTA, 2.5 % glycerol, 1.3 % polyvinylalcohol (polymerization degree about 500), 0.005 % Triton X-100, 1mM DTT) and unbound protein was removed by washing three times with 200 μL of 4E-PD buffer. Prior to the cap-purification, total RNA was first subjected to LiCl-precipitation twice to remove abundant 5' capped small ncRNAs. The LiCl-precipitated RNA (30 μg) was heat-denatured at 65 °C for 10 min and mixed with GST-eIF4Ek119A-bound MagneGST particles in 200 μL of 4E-PD buffer supplemented with 25 μg yeast tRNA and 40 U recombinant RNase inhibitor (TaKaRa) for 2 hr at 25 °C. After washing three times with 500 μL of 4E-PD buffer, GST-eIF4Ek119A was eluted in 100 μL of elution buffer (100 mM Tris-HCl (pH 8.6), 1 mM EDTA, 0.005 % Triton X-100, 150 mM NaCl, 40 mM glutathione, 1 mM DTT, 5 U/mL Turbo3C protease) at r.t. for 10 min. The elution was repeated four times, and the eluate was combined and phenol-chloroform extracted followed by EtOH precipitation in the presence of glycogen (Roche). The precipitated RNA was dissolved in 10 μL of QW and further purified using 1.8 volume of Agencourt RNAClean XP (Beckman). After elution in 10 μL of QW, RNA concentration was determined using QuantiFluor RNA System (Promega). Library preparation for nanopore sequencing was performed using the Oxford Nanopore's direct RNA sequencing kit (SQK-RNA001 or SQK-RNA002) according to the manufacturer's instruction except that we used 1μg of the cap-purified RNA and 0.5 μL of RNA calibration strand (RCS) for adapter ligation. To improve throughput, we performed reverse transcription using SuperScript III (Thermo Scientific) after the adapter ligation. All the procedures above were performed in DNA LoBind tubes (Eppendorf). Nanopore direct RNA-sequencing
GEO
GSM4332032
GEO Accession
GSM4332032