GSM3048828: WT5; Mus musculus; RNA-Seq
Total RNA was isolated from fresh tumor samples.  Fresh tumor samples were placed in Trizol LS reagent and homogenized by vortexing, microgrinding and mechanical dissociation through a syringe. Total RNA isolation was subsequently performed using a RNeasy Mini Kit (Qiagen) following the manufacturer's protocol with chloroform, isopropanol and ethanol washes. mRNA from tumor samples was enriched using oligo(dT) beads. The mRNA was then fragmented randomly in fragmentation buffer, followed by cDNA synthesis using random hexamers and reverse transcriptase. After first-strand synthesis, a custom second-strand synthesis buffer (Illumina) was added with dNTPs, RNase H and Escherichia coli polymerase I to generate the second strand by nick-translation. The final cDNA library was ready after a round of purification, terminal repair, A-tailing, ligation of sequencing adapters, size selection and PCR enrichment.
GEO Accession
GSM3048828