Unknown,Transcriptomics,Genomics,Proteomics

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RNA libraries of sorted CD8+ memory T cells from the spleen and RNA libraries of sorted IELs from the small intestine of C57BL/6J mice

ABSTRACT: Intraepithelilal lymphocytes (IELs) are located at the intestinal barrier where they can offer swift protection against invading pathogens. However, they are kept in a heightened state of activation resembling effector T cells, but without cytokine production or clonal proliferation. They also posses altered metabolic pathways than CD8+ memory T cells from spleen. With differentially expressed gene analysis of intestinal IELs and CD8+ memory T cells from spleen, we confirmed increased expression of metabolic enzymes involved in lipid uptake and lipid metabolism in IELs compared with CD8+ memory T cells. This was particularly the case for enzymes involved in mevalonate, lanosterol and cholesterol synthesis pathways, suggesting increased lipid metabolite generation. Differential gene expression showed also strong predisposition for cytotoxic potential that IELs possess in comparison to CD8+ memory T cell.

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Mus musculus

SUBMITTER: Spela Konjar

PROVIDER: E-MTAB-6510 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:One day-old broilers (Ross/Ross, Longenecker's Hatchery, Elizabethtown, PA) were housed in Petersime starter brooder units and randomly assigned to 4 groups (5 birds/group). Carvacrol (Nature Chemicals, Hamburg, Germany) and cinnamaldehyde (Alys Technologies SA, Bussigny-près-Lausanne, Switzerland) were obtained from the compound obtained from synthesis. Crushed C. annuum fruits (Pushp Brand Spices, Munimji Foods and Spices Pvt. Ltd. Indore, India) were extracted with volatile solvents using the percolation method to obtain oleoresin. Chickens were fed for 7 days beginning from hatch with a standard diet alone (control) or with diets supplemented with carvacrol, cinnamaldehyde, or Capsicum oleoresin.<br> Following euthanization of the birds, intestines were cut longitudinally and washed three times with ice-cold Hanks? balanced salt solution (HBSS) containing 100 U/ml of penicillin and 100 mg/ml of streptomycin (Sigma, St. Louis, MO). The mucosal layer of entire intestine was carefully scraped using a surgical scalpel and intraepithelial lymphocytes (IELs) were isolated by Percoll density gradient centrifugation as previously described (Min et al. 2005). Total RNA was isolated from 5.0 x 107 cells using Trizol (Invitrogen, Carlsbad, CA) and purified using the RNeasy Mini RNA Purification Kit (Qiagen, Valencia, CA). Aminoallyl-labeled RNA from IELs was prepared using the Amino Allyl Message Amp II aRNA Amplification Kit (Ambion, Austin, TX). Two of 20 ?g aliquots of each aminoallyl-RNA sample were fluorescently labeled with AlexaFluor 555 or AlexaFluor 647 (Invitrogen) and labeled RNAs were column-purified using the RNA Amplification Kit (Ambion). RNA concentrations and labeling efficiencies were determined spectrophotometrically.<br> Hybridizations were performed using HybIt hybridization buffer (TeleChem, Sunnyvale, CA) in ArrayIt reaction cassettes at 50°C overnight as described (Kim et al., 2008). After hybridization, the slides were rinsed in 0.5 X SSC, 0.01% SDS at room temperature and washed once for 15 min in 0.2 X SSC, 0.2% SDS at 50°C, 3 times for 1 min in 0.2 X SSC at room temperature, and 3 times for 1 min in distilled water at room temperature. Each sample had a repeated hybridization using the alternate fluorescent dye between the treatment and control.

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RNA libraries of sorted CD8+ memory T cells from the spleen and RNA libraries of sorted IELs from the small intestine of C57BL/6J mice

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