Livers were quickly rinsed in PBS and then rapidly frozen in liquid nitrogen. All samples stored at -80 until RNA preparation.
Growth protocol
Animals housed in 12-hr L/D cycle. Animals fed adlibitum.
Extracted molecule
total RNA
Extraction protocol
Liver chunks were homogenized in Trizol. Target Preparation/Processing for GeneChip® Analysis Isolated total RNA samples were processed as recommended by Affymetrix, Inc. (Affymetrix Genechip® Whole Transcript Sense Target Labeling Assay Manual, Affymetrix, Inc., Santa Clara, CA) In brief, total RNA was initially isolated using TRIzol Reagent (Gibco BRL Life Technologies, Rockville, MD), and passed through an RNeasy spin column (Qiagen, Chatsworth, CA) for further clean up. Eluted total RNAs were quantified (Nanodrop) with a portion of the recovered total RNA adjust to a final concentration of 100ng/ul. All starting total RNA samples were quality assessed prior to beginning target preparation/processing steps by running out a small amount of each sample (typically 25-250ng/well) onto a RNA 6000 Nano LabChip that was evaluated on an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA).Single-stranded, then double-stranded cDNA was synthesized from the poly(A)+mRNA present in the isolated total RNA (typically 100ng total RNA starting material each sample reaction) using the Ambion® WT Expression Kit (Life Technologies, Carlsbad, CA) and random hexamers tagged with a T7 promoter sequence. The double-stranded cDNA is then used as a template to generate many copies of antisense cRNA from an in vitro transcription reaction (IVT) of 16hrs in the presence of T7 RNA Polymerase. 10 ug of cRNA were input into the second cycle cDNA reaction with random hexamers that are used to reverse transcribe the cRNA from the first cycle to produce single-stranded DNA in the sense orientation. The single-stranded DNA sample is fragmented (WT Terminal Labeling Kit, Affymetrix, Inc, Santa Clara, CA) to an average strand length of 70 bases (range 40-70bp) following prescribed protocols (Affymetrix GeneChip® WT Sense Target Labeling Assay Manual). The fragmented single-stranded DNA is subsequently labeled with recombinant terminal deoxynucleotidyl transferase (TdT) and the Affymetrix proprietary DNA Labeling Reagent that is covalently linked to biotin.
Label
TdT
Label protocol
The fragmented single-stranded DNA is labeled with recombinant terminal deoxynucleotidyl transferase (TdT) and the Affymetrix proprietary DNA Labeling Reagent that is covalently linked to biotin
Hybridization protocol
Following the recommended procedure, 2 ug of this fragmented target single-stranded cDNA was hybridized at 45c with rotation for 17 hours (Affymetrix GeneChip® Hybridization Oven 640) to probe sets present on an Affymetrix GeneChip Mouse Gene 1.0 ST array (Affymetrix, 901171). The GeneChip® arrays were washed and then stained (SAPE, streptavidin-phycoerythrin) on an Affymetrix Fludics Station 450 (Fluidics protocol FS450_007).
Scan protocol
Arrays were scanned using the GeneChip Scanner 3000 7G and Command Console Software v. 3.2.3.to produce .CEL intensity files.
Description
ZT16, high fat diet #2
Data processing
These probe cell intensity files (*.CEL) were analyzed in Affymetrix Expression Console software v1.1.1 using the PLIER algorithm to generate probe level summarization files (*.CHP). (Algorithm: PLIER v 2.0; Quantification Scale: Linear; Quantification Type: Signal and Detection P-Value; Background: PM-GCBG; Normalization Method: Sketch-Quantile).
