GSM9241808: Kidney, ACE-Seq; Mus musculus; OTHER
Extraction was carried out using a phenol/chloroform/isoamyl alcohol protocol with minor modifications {Molecular cloning: a laboratory manual. No. Ed. 2. Cold Spring Harbor laboratory press, 1989}. 500 µL of the lysed mouse tissues were transferred to pre-centrifuged (at 20,000g for 1 min) 5PRIME Phase Lock Gel tube (Quanta bio, 10847- 802). In a fume hood, 500 µL of phenol/chloroform/isoamyl alcohol (Sigma-Aldrich, 77617) was added to the phase Lock Gel tube. The aqueous phase was then transferred to a new 1.5 mL tube. 500 µl of 100% isopropanol (MilliporeSigma), 1.7 µl of GlycoBlue (Invitrogen), and 33 µl of 7.5 M ammonium acetate solution (Sigma-Aldrich) were added. The mixture was vortexed and incubated at -20°C for 30 min overnight. The samples were subsequently centrifuged at 16,000g for 30 min at 4°C. Following centrifugation, the samples were washed twice with 1 mL of 70% ethanol (MilliporeSigma). After the final wash and removal of 70% ethanol, the samples were air-dried for 10 min and then resuspended in 46 to 200 µL of Tris buffer pH 8.0 at 55°C for 10 min. If the DNA was not fully dissolved, it was further incubated at 4°C for one to two days. DNA concentration was measured using a Qubit 4.0 Fluorometer (Invitrogen) with the dsDNA HS Assay Kit (Invitrogen). 30 ng of purified mouse gDNA was mixed with 5% of unmethylated λ gDNA spike-in and 5% 5mCpG methylated plasmid spike-in, in a total volume of 50 µL in low TE buffer. This input DNA was then sheared to 350 bp using a Covaris instrument. Each sample was then split three ways to serve as technical replicates, with each treated as a sample starting from a 10 ng input. Libraries were prepared using the NEBNext Enzymatic Methyl-seq kit in accordance with the manufacturer's instructions, with the noted modifications: 5pyC Y-shaped adaptors were used during ligation rather than the EM-Seq adaptors and only bGT was used rather than TET/bGT as detailed above. 2X KAPA HiFi U+ Polymerase (KAPA) was used to amplify the libraries following conversion and then subjected to a 0.8x left-sided SPRI purification, with elution in nuclease-free water to yield final libraries. Final libraries were quantified with a Qubit HS kit (Invitrogen), assessed for quality using an Agilent TapeStation D1000, and sequenced on an Illumina MiSeq.