GSM2341408: FS8; Geospiza fuliginosa; MeDIP-Seq
Genomic DNA from finch red blood cells (erythrocytes) was prepared using the Qiagen DNAeasy Blood and Tissue Kit (Qiagen, Valencia CA). The manufacturer’s instructions for nucleated blood samples were followed, but in the final DNA elution step H2O was used instead of the buffer provided in the kit. Genomic DNA from finch sperm was prepared as follows: collected sperm suspension was adjusted to 100 μl with 1 x Phosphate Buffered Saline (PBS) then 820 μl DNA extraction buffer (50mM Tris pH8, 10mM EDTA pH8, 0.5% SDS) and 80 μl 0.1 M dithiothreitol (DTT) were added and the sample was incubated at 65°C for 15 minutes. Next, 80 μl Proteinase K (20 mg/ml) were added and the sample was incubated on a rotator at 55°C for 2 hours. After incubation, 300 μl of protein precipitation solution (Promega, A795A) were added, then the sample was mixed and incubated on ice for 15 minutes, then spun at 4°C at 13,000 rpm for 20 minutes. The supernatant was transferred to a fresh tube, then precipitated over night with the same volume of 100% isopropanol and 2 μl glycoblue at -20°C. The sample was then centrifuged and the pellet washed with 75% ethanol, then air-dried and re-suspended in 100 μl H2O. DNA concentration was measured using a Nanodrop Spectrophotometer (Thermo Fisher). The MeDIP pools were used to create libraries for next generation sequencing (NGS) at the University of Nevada, Reno Genomics Core Laboratory using 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.
GEO Accession
GSM2341408