title: GSM3671353: Leghorn biological replicate1 individual injected with avian influenza virus [LI1-C]; Gallus gallus; Bisulfite-Seq
library_construction_protocol: DNA from lung tissue was isolated by phenol-chloroform extraction. Libraries were prepared as below. Five μg DNA was sonicated to produce 200-400 bp fragments, followed by end repair with a nucleotide triphosphate mix free of dCTP. Cytosine methylated adapters provided by Illumina (Illumina, San Diego, CA) were ligated to the sonicated DNA according to the manufacturer's instructions for genomic DNA library construction. Adapter-ligated DNA with the length of 320-500 bp was isolated by 2% agarose gel electrophoresis, and sodium bisulfite conversion was performed on it using the MethylEasy Xceed kit (Human Genetic Signatures, NSW, Australia) was used according to the manufacturer's instructions. The 300 ng of bisulfite-converted, adapter-ligated DNA molecules were enriched by 14 cycles of PCR with the following reaction composition: 2.5 U of uracil-insensitive Pfu TurboCx Hotstart DNA polymerase (Stratagene), 5 μL of 10× Pfu Turbo reaction buffer, 25 μM dNTPs, 1 μL of Primer 1.1, and 1 μL of Primer 2.1 (50 μL final). The thermocycling parameters were as follows: 98°C for 2 min, followed by 4 cycles of 98°C for 15 s, 60°C for 30 s, and 72°C for 1 min, ending with incubation at 72°C for 10 min. The reaction products were purified using the MinElute PCR purification kit (Qiagen, Valencia, CA) and then separated by 2% agarose gel electrophoresis and purified by the MinElute gel purification kit (Qiagen, Valencia, CA). DNA library prepared above was sequenced using the Illumina Genome Analyzer II (GA II) according to the manufacturer's instructions. Each sample had two technical replicates.
attributes.tag: GEO Accession
attributes.value: GSM3671353