title: 153
organism: Gallus gallus
description: Protocols: Forty LB and 40 LSL hatchlings were obtained from Lohmann Tierzucht GmbH, Cuxhaven, Germany. Hens were housed individually in metabolism crates starting at 15 or 20 weeks of age. The experimental diets were provided for four weeks before hens were slaughtered at weeks 19 and 24. This experiment had a 2×2×2-factorial arrangement of treatments with hen strain, hen age (19 and 24 weeks, representing before and after the onset of laying), and mineral P supplement (0 and 1 g P/kg of feed). Diets were maize-soybean meal-based, without a phytase supplement, and otherwise composed to contain all nutrients at recommended levels. The P concentration in the plant-based diet, without a mineral P supplement, was about 3 g/kg, equivalent to approximately 1.5 g/kg of NPP. Ten hens per strain and age were sampled, according to the diet treatments (80 hens in total), and each pen served as one experimental unit for all measurements in the statistical analysis. The hens were killed by electronic stunning and exsanguination from 07:00 to 13:00. Jejunum samples approximately 2 cm were collected from approximately 3 cm distal to the duodenal loop. The samples were cut open; the mucosa was thoroughly rinsed with a 0.9% NaCl solution, and scraped for each bird. The small intestinal mucosa (jejunum) samples were immediately frozen in liquid nitrogen and stored at -80 °C until analysis. ATAC-seq libraries were generated using an already-established protocol for cryopreserved nuclei (Buenrostro et al., 2015; Halstead et al., 2020). Samples were collected, slowly frozen, and cryopreserved to keep the viability of cells. Density gradient centrifugation with iodexanol was employed to eliminate contaminating mitochondria. Nuclei were counted using Trypan solution to assess the optimal viable nuclei (>50,000) needed for transposition using Illumina Tagment DNA Enzyme and Buffer Kit (Tn5 Transposase/Tagment DNA Enzyme 1) according to the manufacturer's protocol (Illumina, San Diego, CA, USA). Tween 20 and Digitonin were added to the transposition reaction to increase nuclear permeabilization and efficiency. PCR was performed to amplify transposed fragments using the Illumina unique dual indexes. The libraries were quality control for fragment length distribution on DNA chip using Agilent Bioanalyzer 2100.