Illumina HiSeq 2500 paired end sequencing; RNA-seq of whole ileums from wildtype mice that were either exposed or not with diets with different protein concentrations for 4 weeks
RNA-seq of whole ileums from wildtype mice that were either exposed or not with diets with different protein concentrations for 4 weeks
The mice were sacrificed and the small intestine tissue was dissected, rinsed, and saved in RNAlater for RNA preservation. Mice were housed in an enhanced barrier specific pathogen-free facility under a strict 12-hour light cycle.  Mice were maintained on a C57BL/6 background. All study protocols were approved by the Washington University Animal Studies Committee. Mice were fed irradiated standard diet (PicoLab Rodent Chow 20, Purina Mills; containing 20% protein), isocaloric protein-free diet (Envigo # TD.99328), or isocaloric 6% protein diet (Envigo # TD.90016). Total RNA was isolated from whole lung homogenates with Trizol Reagent according to the manufacturer's protocol. RNA was DNase-treated and purified via Qiagen RNA Purification columns according to the manufacturer's instructions. Total RNA concentration and purity was obtained from an absorbance ratio at 260nm and 280nm. Total RNA quality was then determined by Agilent 2100 bioanalyzer (Agilent Technologies) according to manufacturers recommendations. Library preparation was performed by the Genome Technology Access Center (GTAC) at Washington University. Library preparation was performed with 1ug of total RNA.  Total RNA integrity was determined using an Agilent bioanalyzer. Ribosomal RNA was removed by a hybridization method using Ribo-ZERO kits (Illumina-EpiCentre). mRNA was then fragmented in buffer containing 40mM Tris Acetate pH 8.2, 100mM Potassium Acetate and 30mM Magnesium Acetate and heating to 94 degrees for 150 seconds. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturers instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3 ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12 cycles using primers incorporating unique index tags. Fragments were sequenced on an Illumina HiSeq-2500 using single reads extending 50 bases.