title: GSM8491143: hMe-Seal-seq, GBM33, tumor; Homo sapiens; OTHER
library_construction_protocol: GBM tissue was digested in a lysis buffer (10 mM Tris pH 8.0, 5 mM EDTA, 200 mM NaCl, 0.2% SDS) with 30 l proteinase K (20 mg/ml) and incubated at 55C overnight. After the overnight digestion, the lysates were brought to room temperature and incubated with 5 L of RNase A solution (20 mg/ml) for 1 hour at 37 C. This was followed by adding equal volume of buffered phenol:chloroform:isoamyl alcohol (25:24:1 ratio) and centrifuged at 14,000 RPM at room temperature for 20 minutes. Supernatant was transferred to clean tubes and 5 l of 5 M NaCl, 2 l Glycogen, and equal volume of 100% ethanol were added. After overnight incubation at -20C, samples were centrifuged at 10,000 g for 10 minutes at 4C, and the DNA pellet was washed in 70% ethanol twice. After all ethanol was removed, the DNA pellet was dissolved in nuclease-free water and incubated overnight at 4C, before being quantified by Nanodrop. 5hmC capture was performed according to the methods described in Kuehner et al, 2021. 5 µg of genomic DNA was fragmented to 300-400 base pairs through sonication and 5hmCs were glycosylated by T4 phage ß-glucosyltransferase enzyme and UDP-6-N3-glucose. The glycosylated fragments were purified with Ampure XP beads (BeckmanCoulter; Cat. A63881), biotinylated by click reaction with disulfide biotin, and pulled down with Dynabeads MyOne Streptavidin C1 beads (ThermoFisher; Cat. 65001). The 5hmC fragments were released from the beads using dithiothreitol and purified with Ampure XP beads for a final time. DNA fragments were eluted in nuclease-free water and quantified by Qubit.