GSM9192243: input from flow-sorted chromosome 13; Homo sapiens; OTHER
Chromosomes were mitotically arrested then isolated with flow cytometry. Two rounds of sorting were used to increase purity. Flow cytometry data were collected and analyzed with FACSDiva (BD Bioscience) and FlowJo software (BD Bioscience). Genomic DNA from sorted chromosomes was isolated using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer's protocol. Methyl-seq libraries were generated from 2.4–6.6ng of DNA isolated from sorted chromosomes, as assessed using the Qubit Fluorometer (Life Technologies). Libraries were made according to the manufacturer's directions for the NEBNext Enzymatic Methyl-seq kit (New England Biolabs, Cat. No. E7120S) using the S220 Focused-ultrasonicator (Covaris) to shear to 300bp and 9 cycles of library PCR amplification. Resulting short fragment libraries were checked for quality and quantity using the Bioanalyzer (Agilent) and Qubit Fluorometer (Life Technologies). Libraries were pooled, requantified and sequenced as 150bp paired reads on a mid-output flow cell using the Illumina NextSeq 500 instrument, utilizing RTA and instrument software versions current at the time of processing.