GSM1196912: Adult liver 2_5hmC; Homo sapiens; OTHER
AllPrep DNA/RNA/Protein Mini kit (QIAgen, Cat. # 80004) 1) gDNA samples (3 µg in 120 µl) were sonicated using a Covaris S2 (Duty Cycle = 10%, Intensity = 5, Cycles per Burst = 200, Time = 6 cycles (60s each), Mode = Frequency Sweeping, Temperature = 4°C). Sonicated DNA was purified once with 180 µl of Agencourt AMPure XP beads (Beckman-Coulter, Cat. # A63880); 2) Sheared DNA samples were enriched for the 5hmC-containing fraction with the Hydroxymethyl Collector kit (ActiveMotif, Cat. # 55013) following manufacturer’s instructions. In parallel, non-enriched aliquots of sheared DNA were diluted 1:100; 3) 10 ng of sonicated gDNA (either 5hmC-enriched or non-enriched) were processed with NEBNext End Repair Module (New England Biolabs, Cat. # E6050S) and then purified with MinElute Reaction Cleanup kit (QIAgen, Cat. # 28204); 4) End repaired samples were treated with NEBNext dA-Tailing Module (New England Biolabs, Cat. # E6053S) and then purified with MinElute Reaction Cleanup kit; 5) dA tailed samples were ligated to TruSeq adapters (1.5 ul) using NEBNext Quick Ligation Module (New England Biolabs, Cat. # E6056S). The adapters were diluted 1:50 with 0.1x TE from TruSeq DNA Sample Prep kit v2 (Illumina Inc., Cat. # FC-121-2001); 6) Adapter ligated samples were purified twice with Agencourt AMPure XP beads (50 ul DNA + 30 ul beads); 7) Purified samples were PCR amplified: 37 ul DNA + 1 ul TruSeq primer 1.0 + 1 ul TruSeq primer 2.0 + 0.5 µl dNTPs mix (25 mM each) + 10 µl Herculase II Buffer + 1 µl Herculase II Fusion DNA polymerase (Agilent, Cat. # 600675). Program: 95°C, 5' (1 cycle); 98°C, 30” (1 cycle); 98°C, 10” -> 63°C, 30” -> 72°C, 30” (14 cycles); 72°C, 5’ (1 cycle); 8) Amplified NGS libraries were purified once with Agencourt AMPure XP beads (50 ul DNA + 50 ul beads), quantified with QPCR NGS Library Quantification kit for Illumina GA (Agilent, Cat. #G4880A), pooled and sequenced on Illumina HiSeq2000;
GEO Accession
GSM1196912