GSM4278391: HPVneg9; Homo sapiens; MeDIP-Seq
Using a sterile scalpel, surface scrapings were taken directly from the frozen tissue blocks from the region of tissue identified as having at least 70% tumor cellularity. Tissue scrapings were processed using the Qiagen AllPrep DNA/RNA/Protein Mini Kit (Valencia, CA, USA) as per manufacturer protocol. A total of 1μg of genomic DNA was used for shearing, blunt-end repair and phosphorylation process, and a single adenine nucleotide was then added to the 3' end of the resulting fragments for ligation preparation. DNA was cleaned by Qiagen's MinElute PCR purification columns (Qiagen, Germantown, MD). DNA samples were denatured and resuspended in ice-cold immunoprecipitation buffer after the addition of DNA spike-ins for hMeDIP (Diagenode Denville, NJ). At this stage, 10% volume of the DNA solutions were kept as inputs, and immunoprecipitation overnight at 4°C with rotation was performed on the remaining solution, after adding a 5hmC-specific antibody (Cat # 39791, Active Motif, Carlsbad, CA). The 5hmC-enriched DNA fragments (IP) were released from the antibody and cleaned-up by Proteinase K (ThermoFisher, Carlsbad, CA) and AMPure XP beads (Beckman Coulter, Brea, CA), respectively. In order to evaluate the percent enrichment over input in the IP, qPCR with primers for spike-ins was conducted. For samples with good percent enrichment over input, PCR amplification was performed for library production, followed by cleaning with AMPure XP beads and quantification with the Qubit assay (ThermoFisher, Carlsbad, CA) and TapeStation High Sensitivity D1000 kit (Agilent, Santa Clara, CA).
GEO Accession
GSM4278391