title: GSM6701304: S9_5hmC.19_Normalized_Counts; Homo sapiens; OTHER
library_construction_protocol: DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Cat.#69504) with RNase treatment. DNA concentrations were measured by a spectrophotometer (NanoDrop) and diluted to 100ng/μL with TE buffer. The aliquot (100μL) of genomic DNA was sonicated using a Bioruptor® Pico instrument (Diagenode) for 7-10 cycles at 30 seconds on and 30 seconds off. The size of fragmented DNA was determined using a Fragment Analyzer (Advanced Analytical Technologies, Ankeny, IA) with the High Sensitivity NGS Fragment Analysis Kit (Cat.#DNF-486). Fragmented DNA with an average size of 200bp was denatured for 10min at 95°C, after which 2.5-5μg of DNA in 1X DNA immunoprecipitation (DIP) buffer (10mM sodium phosphate, pH 7.0, 140mM NaCl, 0.05% Triton X-100) was incubated with 1μg of anti-5hmC antibody from the hybridoma clone EDL HMC 1A (equivalent to Cat.#MABE1093 from MilliporeSigma) for 3hr at 4°C on a rotator. Dynabeads Protein-G (Thermo-Fisher, Cat.#10003D) were added to the DNA/antibody mixture, and the reaction was incubated at 4°C overnight on a rotator. Collected beads-antibody-DNA complexes were then thoroughly washed with DIP buffer and TE buffer. The enriched DNA fragments were eluted from the beads, purified using the ssDNA/RNA Clean & Concentrator (Zymo Research, Cat.#D7010), and quantified with the Qubit ssDNA High Sensitivity Assay Kit (Thermo-Fisher Scientific, Cat.#Q10212). DNA libraries were generated from input and enriched DNAs using the Accel-NGS® 1S Plus DNA Library Kit (Swift Biosciences, Cat.#10024), according to the manufacturer's protocol