title: GSM3927401: RFP_EpiLSCs_Rep2; Mus musculus; OTHER
library_construction_protocol: Illumina RNA-seq strand specific Briefly, 500 µg of the ribonucleoproteins (2 biological replicates per sample) were treated with 1000U RNase I (Ambion Cat# AM2295) at 4oC for 50 min with gentle end-over-end rotation followed by incubation with SuperaseIn (Ambion, Cat# AM2696). Monosomes were pelleted by ultracentrifugation in a 34% sucrose cushion at 70,000 rpm in TLA-120.2 rotor (Beckman Coulter) at 4oC for 3h. RNA fragments were extracted with acid phenol (2x), once with chloroform, and precipitated with isopropanol at -20oC in the presence of NaOAc and GlycoBlue (Invitrogen). Purified RNA samples were resolved on a denaturing 15% polyacrylamide-urea gel and the sections corresponding to 28-32 nucleotides containing the ribosome footprints (RFPs) were excised, eluted, and precipitated by isopropanol. Purified RFPs were dephosphorylated using T4 polynucleotide kinase (New England Biolabs) for 1 h at 37°C. Denatured fragments were resuspended in 10 mM Tris (pH 7) and quantified using the Bio-Analyzer Small RNA assay (Agilent). A sample of 10 pmol of RNA was ligated to the 3′-adaptor with T4 RNA ligase 1 (New England Biolabs) for 2 h at 37°C. Reverse transcription was carried out using oNTI223 adapter (Illumina) and SuperScript III reverse transcriptase (Invitrogen) according to the manufacturer's instructions. Products were separated from the empty adaptor on a 10% polyacrylamide Tris/Borate/EDTA-urea (TBE-urea) gel and circularized by CircLigase (Epicentre). Ribosomal RNA amounts were reduced by subtractive hybridization using biotinylated rDNA complementary oligos (Ingolia et al., 2012). The RFP libraries were amplified by PCR (11 cycles) using indexed primers and quantified using the Agilent BioAnalyzer High-Sensitivity assay. DNA was then sequenced on the HiSeq-2000 platform with read length of 50 nucleotides (SR50) according to the manufacturer's instructions, with sequencing primer oNTI202 (5CGACAGGTTCAGAGTTCTACAGTCCGACGATC).
identifiers.namespace: GEO
identifiers.id: GSM3927401