title: GSM2840013: R98S13_RiboSeq; Mus musculus; OTHER
library_construction_protocol: Ribosome footprinting libraries. RNA fragments  were purified using Trizol. RNA-Seq libraries. RNA was isolated using the Maxwell 16 LEV simplyRNA Cells Kit (Promega) Ribosome footprinting libraries. RNA was gel-purified on a denaturing 10% polyacrylamide urea (7 M) gel. A section corresponding to 30 to 33 nucleotides was excised, eluted and ethanol precipitated. The resulting fragments were 3′-dephosphorylated using T4 PNK (New England Biolabs) for 4 hrs at 37°C in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (100 mM MES-NaOH, pH 5.5, 10 mM MgCl2, 10 mM β-mercaptoethanol, 300 mM NaCl). 3′ adaptor was added with T4 RNA ligase 1 (New England Biolabs) for 2.5 hrs at 37°C. Ligation products were 5′-phosphorylated with T4 polynucleotide kinase for 30 min at 37°C. 5′ adaptor was added with T4 RNA ligase 1 for 18 hrs at 22°C. To deplete ribosomal RNAs (rRNA), the resulting fragments were incubated with a biotinylated rRNAs oligo pool (10 μM each) for 10 min at 37°C, upon short denaturation at 95-100 °C for 1 min. rRNA depletion was carried on using MyOne Streptavidin C1 DynaBeads (ThermoFisher Scientific). The resulting rRNA-depleted fragments were reverse transcribed using the SuperScript III cDNA synthesis kit. Samples were PCR-amplified for 15 cycles and the products were multiplexed and sequenced on an Illumina HiSeq 2000 platform in single read 50 nucleotides mode. RNA-Seq libraries. Libraries were generated from total RNA using the TruSeq Stranded mRNA Sample Prep Kit (Illumina)
attributes.tag: GEO Accession
attributes.value: GSM2840013