GSM3732569: EOD_rep2 [EOD_3M2]; Mus musculus; RNA-Seq
The left lateral liver lobe was then harvested and immediately flash frozen before being stored at -80°C RNA samples from the liver was isolated using EZ-10 DNAaway RNA Mini-Preps Kit (Bio Basic, Canada) according to the manufacturers' protocol. Briefly, frozen liver samples were homogenized and lysed in the provided lysis buffer. Prevention of genomic DNA contamination was achieved using the provided gDNA eliminator column. The RNA's purity was determined by using the Nanodrop ND-1000 (Thermo Fisher Scientific, USA), whereas the RNA's integrity was assessed via agarose gel electrophoresis as well as from the Agilent 2100 Bioanalyzer (Agilent, USA). Enrichment of high-quality RNA samples should demonstrate an OD260/OD280 ratio of 1.9-2.0 from Nanodrop readings, two distinct bands indicating 28S and 18S following agarose gel electrophoresis, as well as showing ≥ 6.8 RNA integrity number with a smooth base line using the Agilent 2100 Bioanalyzer. Following the isolation of these high-quality and pure total RNA samples from the liver tissues, cDNA library construction was mediated using the NEBNext® UltraTM RNA library preparation kit as per the manufacturers' protocol (New England BioLabs, USA). mRNA was first purified via the addition of poly-T-oligo-attached magnetic beads, before subjecting them to random fragmentation mediated by the usage of a fragmentation buffer. The initial strand of the cDNA is first synthesized through the utilisation of a random hexamer primer and RNase H- (M-MuLV reverse transcriptase). The second strand of cDNA is then synthesized using DNA polymerase I and RNase H, and the resulting double stranded cDNA is then purified using AMPure XP beads. The overhangs of these purified double-stranded cDNA were then processed by exonuclease and polymerase to become blunt ends, before subjecting the 3' ends of these DNA fragments to adenylation and subsequent ligation of NEBNext hairpin loop structure adaptor on both ends for hybridisation. For optimal isolation of cDNA fragments of approximately 150-200 base pairs in length, DNA fragments were then purified using the AMPure XP system (Beckman Coulter, USA), before utilising both PCR amplification and purification by AMPure XP beads to obtain the completed library DNA fragments. The resultant libraries were then sequenced using HiSeqTM 2500 Illumina platforms to obtain 12GB of raw data per sample (Illumina, USA). RNA libraries were prepared for sequencing using standard Illumina protocols
GEO Accession
GSM3732569