alias: GSM3679617
title: GSM3679617: Input_DMSO_1; Homo sapiens; RNA-Seq
library_construction_protocol: Cells were washed once in ice-cold PBS supplemented with 100 µg/ml CHX and harvested by scraping in polysome lysis buffer (20 mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 mg/ml CHX, 1% Triton X-100, 40 U/ml RNasin, EDTA-free complete protease inhibitors (Roche)). Lysates were incubated end over end for 10 min at 4°C and cleared by centrifugation at 10,000 rpm for 10 min at 4°C. The DMSO and Ro3306 treated sample was adjusted to the same OD260 and afterwards combined to one sample. Yeast polysome lysate (2% of the RPE1 lysate) was spiked in. 10% of the lysate was saved as input sample. The lysate was subsequently digested with RNase I (60 units/ per OD260) for 5 min at 4°C. The reaction was stopped by the addition of Superase Inhibitor (6 units/per sample). Samples were loaded onto linear 17 to 50% sucrose gradients (dissolved in 20 mM Tris-HCl pH 7.5, 5 mM MgCl2, 150 mM NaCl). Sucrose density gradient centrifugation was carried out at 35,000 rpm at 4°C using a SW60 rotor (Beckman) for 2.5 h. Sucrose gradients were fractionated by time (14 s/ fraction) using a Teledyne ISCO Foxy Jr. system.  RNA was purified from the cytoplasmic lysate (input) or from the monosomal fractions (ribosome protected fragments)  with Phenol:Chloroform:Isamylalcohol (25:24:1) by phase separation.  Both input and ribosome protected fragements were depleted of rRNA with the Ribo-Zero Gold Kit (Illumina).  Input RNA was randomly fragmented at pH ~10 for 12 min at 95°C.  Fragmented RNA and ribosome protected fragments were size-selected (25 - 35 nt) on a 15% polyacrylamide TBE-urea gel.  After end-repair with T4 PNK, 3 ng per sample were used for library preparation. Small RNA libraries were prepared with the NEXTflex Small RNA-Seq Kit v3 according to the manufacturer's manual.
identifiers.namespace: GEO
identifiers.id: GSM3679617