Qubit fluorimetry

Frozen cell pellets were thawed and lysed using QIAshredder spin columns (Qiagen 79656). Genomic DNA was extracted from each sample using the AllPrep DNA/RNA Mini Kit (Qiagen 80204), then stored at −80 °C before analysis. DNA was quantified by Qubit fluorimetry (Life Technologies). Approximately 500 ng of genomic DNA was bisulfite converted using the Zymo EZ DNA methylation kit (Zymo Research D5004) then hybridized overnight on an Infinium MethylationEPIC BeadChip (Illumina), in which the genomic DNA molecules anneal to locus-specific DNA oligomers linked to individual bead types. Raw signal intensities were exported as.idat files, which were processed using the R package SeSAMe^{63 (link),64 (link)}. Of 386 DNA methylation samples run, 14 failed quality control and were excluded from further analysis, producing a final analytical sample count of 372. All DNA methylation data can be accessed through the Gene Expression Omnibus (GEO) accession GSE197512.

DNA methylation was evaluated using the Illumina HumanMethylationEPIC (EPIC) array. The EPIC platform analyzes the DNA methylation status of up to 863,904 CpG loci and 2,932 non-CpG cytosines, spanning gene-associated CpGs and a large number of enhancer/regulatory CpGs in intergenic regions^{67 (link)}. Briefly, DNA was quantified by Qubit fluorimetry (Life Technologies), and 500 ng of DNA from each sample was bisulfite converted using the Zymo EZ DNA methylation kit (Zymo Research) following the manufacturer’s protocol using the specified modifications for the Illumina Infinium methylation assay. After conversion, all bisulfite reactions were cleaned using the Zymo-Spin binding columns and eluted in Tris buffer. Following elution, bisulfate-converted DNA was processed through the EPIC array protocol. For FFPE samples, the entire bisulfate-converted eluate was used as input for the Infinium HD FFPE DNA Restore kit and processed through the separate restoration workflow. To perform the assay, converted DNA was denatured with NaOH, amplified and hybridized to the EPIC bead chip. An extension reaction was performed using fluorophore-labeled nucleotides per the manufacturer’s protocol.

The sheep were euthanized with an overdose (15 mg/kg) of sodium pentobarbital (Euthozol; Delmarval Laboratories Inc, Midlothian, VA). The medial basal hypothalamus was dissected as a block of tissue that extended from the caudal aspect of the optic chiasm to the rostral aspect of the mammillary bodies, bilaterally to the optic nerves and dorsally to the top of the third ventricle. The dissection was split through the ventricle into left and right halves that were frozen immediately on dry ice and stored in a -80°C freezer. Genomic DNA was extracted from one half of the hypothalamus using the DNeasy Blood & Tissue Kit (Qiagen, Germantown, MD, USA) and concentrated using the Genomic DNA Clean & Concentrator Kit (Zymo Research, Irvine, CA, USA) as directed by the manufacturer. The concentration and quality of genomic DNA was verified with absorbance spectroscopy and Qubit fluorimetry (ThermoFisher Scientific, Waltham, MA, USA). RNA was extracted from the remaining half of the hypothalamus using the RNeasy Mini kit (Qiagen). RNA was quantified with the Qubit RNA Broad range kit (Thermofisher) and integrity was verified on a 4200 Tape station (Agilent, Santa Clara, CA, USA). All RNA samples that were used in these studies had RIN values greater than 8.0.

Genomic DNA was extracted from mid-logarithmic phase culture following the method of Sokolov et al. [64 (link)] with some modifications: sample lysis was performed with a buffer containing 50 mM NaCl, 50 mM Tris-HCl pH8, 50 mM EDTA, 2% SDS, and isopropanol precipitation was replaced by SPRI bead purification. The quality and quantity of isolated DNA were assayed using 1% (w/v) agarose gel electrophoresis and Qubit fluorimetry (Thermo Scientific, Waltham, MA, USA), respectively. The Illumina sequencing library was constructed using the NEBNext Ultra DNA kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. The genome of CCB-ST2H9 was sequenced on a NovaSeq 6000 (Illumina, San Diego, CA, USA) at 150 bp paired-end, generating 10.26 million reads totaling 1.53 Gb. The DNA library for Nanopore sequencing was prepared using the Ligation sequencing kit (SQK-LSK109, Oxford Nanopore, UK). The library was sequenced with a PromethION and yielded 182,476 reads with a total of 1.92 Gb.

Approximately 5,000-10,000 animals were washed off of NGM plates in M9 buffer and allowed to settle to the bottom of a 15 mL conical tube by gravity. After removing the supernatant the worms were washed with 10 mL of fresh M9 and again allowed to settle to the bottom of the tube. All but 1-2 mL of buffer were removed and used to resuspend the worms before transferring them to cryovials where more buffer was removed, leaving behind sufficient volume to cover the worm pellet. After adding 300 μL of Trizol reagent (ThermoFisher Scientific, Waltham, MA), the worms were vortexed in a series of 30 sec intervals over a period of 5 min interspersed by brief rest periods and then transferred to -80° for storage. To prepare total RNA from worm pellets, frozen animals in Trizol were thawed at room temperature and then briefly vortexed before pelleting in a microcentrifuge at 16,000xg for 5 min to remove worm carcasses and debris. Following a phenol/chloroform extraction, total RNA was precipitated in isopropanol, washed in 70% ethanol, and resuspended in RNase-free H₂O. For RNAseq experiments, total RNA was treated with DNase and reisolated over a column using the RNeasy kit (Qiagen, Germantown, MD). The concentration of RNA was measured by either Nanodrop (ThermoFisher Scientific, Waltham, MA) or by Qubit fluorimetry (Invitrogen, Carlsbad, CA).