title: GSM9477285: Kidney, ACE-Seq, rep2; Mus musculus; OTHER
library_construction_protocol: Genomic DNA from different tissue types (cerebellum, heart, kidney, blood) were extracted from C57BL/6J mice (Jackson Labs). Extraction was carried out using a phenol/chloroform/isoamyl alcohol protocol. 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). For shallow-sequenced mouse tissues, 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 ACE-Seq, EM-Seq, Integrated ACE-Seq, or Integrated EM-Seq protocols detailed in the corresponding manuscript. Final libraries were quantified with a Qubit HS kit (Invitrogen), assessed for quality using an Agilent TapeStation D1000, and sequenced on an Illumina MiSeq. For deeply sequenced samples, the same protocol was used except with 50 ng of input used, without splitting.