GSM4261492: 2017_553b: C2-Astro-GFP technical replicate 2; Mus musculus; RNA-Seq
Polysomes were extracted using our recently published TRAP protocol (Boulay AC et al., Methods Mol Biol. 2019). In order to avoid nonspecific binding of the mRNAs to the beads or to IgG, and in contrast to the initial TRAP protocol (Heiman M et al., Nat Protoc. 2014), the cytosolic extracts were pre-cleaned more extensively prior to immunoprecipitation. Lysates were first placed on an antibody-free pre-cleaning column and then on a column with bound nonspecific mouse IgGs. Nonspecific sites on the immunoprecipitation column were blocked with yeast tRNA and BSA. Polysomal mRNAs were purified using the RNeasy Lipid tissue kit (Qiagen). 10 ng of total RNA were amplified and converted to cDNA using SMART-Seq v4 Ultra Low Input RNA kit (Clontech). Afterwards an average of 150 pg of amplified cDNA was used to prepare library following Nextera XT DNA kit (Illumina)
GEO Accession
GSM4261492