GSM1604100: WT-st rep1; Mus musculus; ncRNA-Seq
Ribosome footprint was performed according to (Ingolia et al, 2011). 2x107 primary haematopoietic cells from each genotypes were treated with cycloheximide (100 µg/ml) for 5 minutes prior to lysis in 200 µl of 15 mM Tris-HCl (pH 7.4), 15 mM MgCl2, 300 mM NaCl, 1% Triton X-100, 0.1% 2-Mercaptoethanol, 100 µg/ml cycloheximide, 500 µg/ml Heparin, 1x Complete Protease Inhibitors (Roche). 400 µl aliquot of lysate was treated with 4 U DNase I (ThermoScientific) and 800 U of RNase I (Ambion), for 45 minutes at room temperature with gentle shaking.  800 U of RNasin ribonuclease inhibitor (Promega) was added to quench the reaction. The  samples were run on a 17.5-50% sucrose gradient to isolate monosome. Footprint monosome RNA was end using RNA were end-repaired with T4 polynucleotide kinase (TaKaRa), and size selected 28-31 nucleotide on a 15% polyacrylamide TBE-urea gel. Small RNA libraries were performed according to the protocol using NEB NEXT Small RNA library Prep Set for Illumina (Multiplex Compatible) E7330, and size-selected (150 base pairs) before sequencing.
GEO Accession
GSM1604100