GSM3505044: treEb_1; Mus musculus; RNA-Seq
Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's procedure. The total RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number >7.0. Approximately 10 ug of total RNA was subjected to isolate Poly (A) mRNA with poly-T oligoattached magnetic beads (Invitrogen). Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were reverse-transcribed with Random Hexamers to create the cDNA library, and then synthesized the second strands with addition of buffer, dNTPs, RNaseH and DNA polymerase to be double-stranded DNA. Double-stranded DNA fragments with overhangs were repaired to have blunt ends by Klenow DNA polymerase and T4 DNA polymerase and then added A-overhang as well as adapters before purified by AMPure XP beads. Purified DNA fragments were amplified by PCR before purification again to generate the final library for sequencing. The paired-end sequencing was performed on an Illumina Hiseq4000 following the vendor's recommended protocol and generated reads of 2x150bp (150 PE)
GEO Accession
GSM3505044