GSM8726392: Liver, HFD, Total Knock out, rep1; Mus musculus; RNA-Seq
RNA was extracted using QIAGEN RNeasy Mini Kit (50) kit following manufacturer's instructions Samples were quantified on a NanoDrop ND-1000 instrument. Agarose electrophoresis was used to check the integrality of total RNA samples. 1~2μg total RNA of each sample was taken for RNA-seq library preparation. Briefly, mRNA was isolated from total RNA with NEBNext® Poly(A) mRNA Magnetic Isolation Module. Alternatively, rRNA is removed from the total RNA with a RiboZero Magnetic Gold Kit; The enriched mRNA or rRNA depleted RNA was used for RNA-seq library preparation using KAPA Stranded RNA-Seq Library Prep Kit (Illumina). The library preparation procedure included: 1.1 Fragmentation of the RNA molecules; 1.2 Reverse transcription to synthesis first strand cDNA; 1.3 Second strand cDNA synthesis incorporating dUTP; 1.4 End-repair and A-tailing of the double stranded cDNA; 1.5 Illumina compatible adapter ligation; 1.6 PCR amplification and purification for the final RNA-seq library. The completed libraries were qualified on Agilent 2100 Bioanalyzer for concentration, fragment size distribution between 400~600 bp, and adapter dimer contamination. The amount was determined by absolute quantification qPCR method. The barcoded libraries were mixed in equal amounts and used for sequencing on the instrument.