title: Illumina HiSeq 2500 sequencing; RNA-seq of full thickness small intestine from Atg16l1 T300A and wildtype littermate mice that were either exposed or not with mainstream cigarette smoking for 6 weeks
design: RNA-seq of full thickness small intestine from Atg16l1 T300A and wildtype littermate mice that were either exposed or not with mainstream cigarette smoking for 6 weeks
library_construction_protocol: Mice were housed in an enhanced barrier specific pathogen-free facility under a strict 12-hour light cycle and fed irradiated chow diet (PicoLab Rodent Chow 20, Purina Mills). Mice were maintained on a C57BL/6 background. A heterozygous x heterozygous breeding scheme was used to generate Atg16l1 T300A and wildtype littermate control mice. All study protocols were approved by the Washington University Animal Studies Committee. Four- to six-week-old mice were exposed to mainstream cigarette smoking 4 cigarettes/day, 5 days/week, for 6 weeks, using Kentucky research cigarette 3R4F in a smoking chamber. All mice were sacrificed after completing 6 weeks of smoking exposure, and full thickness ileum were stored in RNAlater for RNA extraction. RNA was isolated using Qiagen RNeasy Minikit, according to the kit protocol. Total RNA concentration and purity was obtained from an absorbance ratio at 260nm and 280nm. Total RNA was quantified using the Quant-iT™ RiboGreen® RNA Assay Kit and normalized to 4ng/ul.  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 manufacturer's 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.