title: GSM4774088: Pm23; Poecilia mexicana; MeDIP-Seq
library_construction_protocol: Fish were sacrificed by cervical transection, and blood was immediately extracted by using microhematocrit capillary tubes (Fisher). Tubes were then spun down at 12,000 rpms for 3 minutes using a ZipCombo Centrifuge (LW Scientific) to separate blood cells from serum. After centrifugation, capillaries were broken to retain only red blood cells. The portion of the capillary containing the red blood cells was placed in a 1.5 ml Eppendorf tube for DNA extraction. DNA was extracted using the Qiagen DNeasy Blood & Tissue kit according to manufacturer's instructions. Extracted genomic DNA was used to isolate methylated DNA with a methyl-cytosine antibody precipitation procedure (MeDIP).  Five micrograms of total genomic DNA was sonicated using a Covaris sonicator. Sonicated DNA was diluted with TE buffer to 400 μl, heat denatured for 10 minutes at 95˚C, and cooled on ice for 10 minutes to create single-stranded DNA fragments. A total of 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the fragmented single-stranded DNA. The mixture was incubated on a rotator overnight at 4˚C. Pre-washed magnetic beads (50 μl, Dynabeads M-280 Sheep anti-Mouse IgG; Life Technologies 11201D) were added to the 500 μl of DNA-antibody mixture. The mixture was incubated for 2 hours on a rotator at 4˚C. After which the samples were washed three times with 1X IP buffer. Washed samples were resuspended in 250 μl digestion buffer (5mM Tris PH 8, 10.mM EDTA, 0.5% SDS) with 3.5μl Proteinase K (20mg/ml) and incubated at 55˚ for three hours. A Phenol-Chloroform-Isoamyalcohol extraction was used to clean-up the DNA and the DNA was resuspended in 20μl of H2O. DNA concentration was measured in Qubit (Life Technologies) with the ssDNA kit (Molecular Probes Q10212). For library preparation, we used the NEBNext Ultra RNA Library Prep Kit for Illumina starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each individual received a unique barcode.