GSM2043500: yes_kcl4_TTAGGGACC_RF; Mus musculus; OTHER
Cell lysate was treated with DNase and clarified, and a portion was taken for RNAseq analysis. Remainder was treated with RNaseI(100U/ul; Invitrogen)for 45 min at room temperature and gentle mixing on a Nutator followed by inactivation of RNase by treatment with SUPERase-In(Thermo Fisher). Ribosomes and protected mRNA fragments were separated from free RNA by pelleting with ultracentrifugation on 1M Sucrose cushion at 200,000g for 4 h at 40C. RNA was isolated from the pellet using miRNeasy kit from Qiagen. Ribosome protected fragments (26-nt to 34-nt size)were purified on a 15% polyacrylamide TBE-urea gel and after dephosphorylation linker ligation was performed at room temperature for 2.5h using miRNA cloning linker from NEB. Linker ligated product was separated from unligated product on a 15% polyacrylamide gel. Reverse transcription was performed using SuperScript III(Invitrogen)per the manufacturer’s protocol. Left over RNA was hydrolysed by treatment with 1N NaOH and precipitated cDNA was circularized using CircLigase(NEB). Circularized cDNA was subjected to rRNA depletion using biotinylated subtraction oligo pool directed against rRNA. Ribosome depleted product was PCR amplified using Phusion polymerase(NEB)and different indexing primers and the final product was purified on 8% polyacrylamide non-denaturing gels. Resuspended library was quantified and fragment size measured on high-sensitivity DNA chip on the Agilent BioAnalyzer.
GEO Accession
GSM2043500