title: GSM8491151: MeDIP-seq, GBM43, tumor; Homo sapiens; MeDIP-Seq
library_name: GSM8491151
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. 3 µg of genomic DNA was fragmented to 300-400 base pairs using a Covaris focused-ultrasonicator Me220. DNA fragments were then subjected to end repair, A-tailing, adaptor ligation, and USER digestion using the NEBNext Ultra II DNA Library Prep kit for Illumina (New England BioLabs, Cat. E7645S), according to the manufacturer's protocol. Following USER digestion and purification, DNA was denatured for 10 minutes at 95C and immunoprecipitated overnight at 4C with 4 L of either 5mC antibody (Active Motif, Cat. 39649) or IgG antibody (Sigma, Cat. 12-371) in IP buffer (500 mM Tris-HCl, pH 7.4, 750 mM NaCl and 0.25% TritonX). The mixture was then incubated with Protein G coated Dynabeads (ThermoFisher, Cat. 10004D) for at least 2 hours at 4C, washed with ice cold IP buffer, and finally washed in ice cold high salt IP buffer (IP buffer plus extra 300 mM NaCl). After the final washing, the beads were treated with 200 L of digestion buffer (1X TE Buffer, pH 7.4, 0.25% SDS, 0.25% Proteinase K (2.5 mg/mL)) and shaken at 1000 rpm for 2 hours at 55C. The methylated DNA was recovered by phenol:chloform:isoamyl alcohol (25:24:1) extraction, followed by precipitation in 3X volume of 100% ethanol, supplemented with 3 L glycogen (5mg/mL) and 15 L NaAC (pH 5.2), and then incubated overnight at -20C. The next day, DNA was pelleted and washed with 75% ethanol and dissolved in nuclease-free water.
identifiers.namespace: GEO
identifiers.id: GSM8491151_r1