GSM5082430: WT_Ctrl_RPF_rep1; Homo sapiens; RNA-Seq
Cells grown in 10-cm dishes were washed once with ice-cold PBS containing 100 µg/ml cycloheximide (CHX) and directly lysed using 290 µl of Ribo-Seq lysis buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 200 mM KCl, 2 mM DTT, 100 µg/ml CHX, 1% NP-40, 40 U/ml RNasin (Promega), EDTA-free complete protease inhibitors (Roche)). The total RNA concentrations between samples were adjusted by measuring absorbance at 260 nm. From the adjusted cellular lysates, 50 µl was taken and diluted with in 250 µl of Ribo-Seq lysis buffer for preparing input samples to be analyzed by RNA-Seq. The remaining lysate (approximate volume of 250 µl) was treated with 240 U/1 A260 of RNase I (Ambion) for 5 min at 4°C on a rotator, and then loaded on top of 3.95 ml 17.5–50% sucrose density gradients  for which sucrose was dissolved in a buffer containing 20 mM Tris-HCl, pH 7.5, 5 mM MgCl2, and 150 mM NaCl. Centrifugation and profile recording were carried out as in the above section, and fractions of 300 µl each were collected with the fraction collector as part of the Teledyne ISCO Foxy RI system. Fractions corresponding to monosomes and disomes were used for preparation of ribosome protected fragments (RPF). Input samples and the monsome/disome fractions were first mixed with 300 µl of urea buffer (10 mM Tris-pH 7.5, 350 mM NaCl, 10 mM EDTA, 1% SDS, 7% urea), incubated with 300 µl phenol:chloroform:isamylalcohol (25:24:1) for 10 min at 65°C, and the RNA was extracted upon centrifugation by recovery of the aqueous phase followed by precipitation using 600 µl isopropanol and 1 µl of GlycoBlue (Invitrogen) at -20°C over night. After washing with 70% ethanol, the RNA was dissolved in 10 µl H2O. To prepare monosome footpints, cell lysates were first separated on 17.5-50% sucrose gradients and monosome fractions were collected manually. Monosome containing fraction was treated with 24 U/1 A260 (according to input sample) of RNase I as indicated above and RNA was purified with the same method with total foot prints. From both input and ribosome protected fragments, ribosomal RNA was depleted from the RFP samples using the Ribo-Zero Gold Kit (Illumina).  Input RNA was randomly fragmented by alkaline hydrolysis at pH 10.0 for 12 min at 95°C. Fragmented RNA and RPFs were then size-selected (25–35 nt) on a 15% polyacrylamide Tris-borate-EDTA-urea gel. The gel pieces were crashed and RNAs were extracted by incubating overnight in 300 µl buffer containing (0.3 M NaCl and 80 u RNase OUT (invitrogen)) at 4oC. Gels were removed by centrifugation at 14.000 x g for 5 min at 4oC with a 0.45 µm NanoSep MF tubes (PALL) and RNAs were precipitated with 300 µl isopropanol and 1 µl GlycoBlue (Ambion). After washing with 70% Ethanol, RNA were dissolved in 14 µl H2O and subjected to end-repair with T4 polynucleotide kinase (NEB) for 1 h at 37°C. Following an additional round of purification and precipitation using isopropanol, upto 20 ng RNA were used for library preparation using the NEXTflex Small RNA-Seq Kit v3 (PerkinElmer) according to the manufacturer's manual. Libraries were multiplexed and sequenced with a NextSeq500 sequencer (Illumina).
GEO Accession
GSM5082430