Instrument: Illumina HiSeq 2500
Strategy: Bisulfite-Seq
Source: GENOMIC
Selection: Reduced Representation
Layout: PAIRED
Construction protocol: To collect blood, animals were placed headfirst into a plastic restraining cone (decapicone, Braintree Scientific) such that the tail remained accessible outside of the cone. The tail was then positioned on a benchtop, ventral side up. A sterile razor blade was used to make a superficial incision across the ventral surface of the tail about halfway between the body and end of the tail. The incision induced bleeding from the tail vein. The animal was then positioned above a blood collection tube (BD Microtainer, tubes with K2EDTA, Becton, Dickinson and Company) so that blood drops from the incision were collected into the collection tube. The collected blood was immediately snap-frozen. After the blood collection, gentle pressure was applied to the tail incision site for one to two minutes to stop further bleeding. The blood DNA was extracted by DNeasy Blood & Tissue Kit (Qiagen 69506) and cleaned up using RNase treatment followed by concentration. The DNA was eluted from Qiagen columns in 100 µl of 10 mM Tris-HCl buffer, pH 8.0. RNA removal was performed with 2 µl of RNase A (Life Technologies) at room temperature for 2 minutes and isolated genomic DNA was purified using Genomic DNA Clean & Concentrator™-10 (Zymo D4011), eluted in 25 µl of 10 mM Tris-HCl buffer, pH 8.0, and quantified using a Qubit 2.0 (Life Technologies AM2271). RRBS libraries were prepared from 100 ng of purified DNA per sample following a protocol described in Petkovich et al. 2017. The libraries were sequenced with Illumina HiSeq2500, PE150. 20% of PhiX genomic DNA was spiked to compensate for the low complexity of the libraries.