title: GSM2463661: S1_Lytle; Potamopyrgus antipodarum; MeDIP-Seq
library_construction_protocol: Genomic DNA from snail foot tissue was prepared as follows. Snail tissue was suspended in 100 μl of 1 x Phosphate Buffered Saline (PBS), then 820 μl DNA extraction buffer (50mM Tris pH8, 10mM EDTA pH8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) was added and the sample incubated on a rotator at 55 C for 2-3 hours. After incubation, 300 μl of protein precipitation solution (Promega, A795A) was added, the sample 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 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 resuspended in 100 μl H2O. Genomic DNA from ten individuals was pooled into a single sample. Three pooled samples were processed for each sampling location.   Methylated DNA Immunoprecipitation (MeDIP) from genomic DNA was performed as follows. For the snail samples approximately 6 μg of each genomic DNA pool was diluted to 130 μl with TE buffer into the appropriate Covaris tube. Covaris was set to 300 bp program. 10 μl of each sonicated DNA was run on 1.5% agarose gel to verify fragment size. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube and the volume measured. The sonicated DNA was then diluted with TE buffer (10mM Tris HCl, pH7.5; 1mM EDTA) to 400 μl, heat-denatured for 10min at 95˚C, then immediately cooled on ice for 5min. Then 100μl of 5X IP buffer and 5μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4˚C.   The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube The same volume of Washing Buffer (at least 1 mL) was added and the bead sample was resuspended. Tube was then placed into a magnetic rack for 1-2 minutes and the supernatant discarded. The tube was removed from the magnetic rack and the washed beads were resuspended in the same volume of 1xIP buffer as the initial volume of beads. 50μl of beads were added to the 500μl of DNA-antibody mixture from the overnight incubation, then incubated for 2h on a rotator at 4˚C.  After the incubation the beads were washed three times with 1X IP buffer as follows: The tube was placed into magnetic rack for 1-2 minutes and the supernatant discarded, then washed with 1xIP buffer 3 times. The washed beads were then resuspended in 250μl digestion buffer (50 mM Tris-HCI pH8, 10mM EDTA pH8, 0.5% SDS) with 3.5μl Proteinase K (20mg/ml). The sample was then incubated for 2-3 hours on a rotator at 55˚C. After incubation the tube was put again into the magnetic rack for 3 minutes and the supernatant removed to a new microfuge tube. The beads were discarded. 250μl of buffered Phenol-Chloroform-Isoamylalcohol solution were added to the supe and the tube vortexed for 30 sec then centrifuged at 14,000rpm for 5min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250μl chloroform were added to the supernatant from the previous step, vortexed for 30sec and centrifuged at 14,000rpm for 5min at room temperature.  The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in  -20˚C freezer for 1 hour to overnight.   The precipitate was centrifuged at 14,000rpm for 20min at 4˚C and the supernatant removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20˚C freezer for 15 min. then centrifuged again at 14,000rpm for 5min at 4˚C and the supernatant discarded. The tube was spun again briefly to collect residual ethanol at bottom of tube and then as much liquid as possible was removed with gel loading tip. Pellet was air-dried at RT until it looked dry (about 5 minutes) then resuspended in 25μl H2O or TE. DNA concentration was measured in Qubit with ssDNA kit. 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 pool received a separate index primer. Next generation sequencing (NGS) was performed at the WSU Spokane Genomic Core laboratory using the Illumina® HiSeq 2500 with a PE50 application, with a read size of approximately 50bp and approximately 100 million reads per pool.  Three libraries each were run in one lane comparing river and lake.
attributes.tag: GEO Accession
attributes.value: GSM2463661