Ptms unlocks chromatin, Fig 7
Brain glial progenitor cells in culture spontaneously transform into neoplastic cells with tumorigenic potential. Transcript analysis identified a histone binding protein up regulated during this process. An examination of its function when introduced into normal cells revealed a dramatic affects on chromatin, with decreased DNA methylation and induction of glioma identity genes. These files contain individual fasta genomic sequences of rat brain glial cells (PC, files djt69), and human embryo kidney (HEK, files djt70, djt86) transfected with a plasmid expression vectors then treated with Ct conversion reagent (Zymo Research, Cat #D5001-1) to distinguish methylated and non-methyl CpG sites. 69-1 OPC x eGFP 69-2 OPC x Ptms 69-3 OPC x H1a 69-4 OPC x H1a,Ptms 70-2 HEK x Ptms 70-3 HEK x H1a 70-4 HEK x H1a Ptms 86-1 HEK x eGFP 86-2 HEK x Ptms 86-3 HEK x H1a 86-4 HEK x H1a, Ptms
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Rutgers U Waksman Genomics Core Facility: Genomic DNA was treated with CT Reagent (Zymo Research; Cat # D5001-1); sequence analysis demonstrated 100% conversion of cytosines to thymidine for the unpaired (non-CpG) cytosines. Target specific (Gfap, CNPase) primers were used to amplify (i) methyl-protected (CpG), and (ii) non-methylated (CpG-to-TpG) targets (Table S1), and the PCR products were cleaned with AMPure XP beads (Beckman Coulter, Inc), concentrations measured with the Qubit dsDNA and Qubit 2.0 Fluorometer (Thermo Fisher), and size distribution verified using an Agilent BioAnalyzer. Libraries were prepared by ligating y-tail sequencing adapters to 50-100 ng template following the TruSeq Nano protocol (Illumina Inc), amplifying each for 5-8 cycles with Illumina TruSeq PCR cocktail, and purifying the products with AMPure XP beads. A BioAnalyzer assay (Agilent) was used to verify ligation and library size, Kapa q-PCR (Kapa Biosystems) was used to determine concentration, and equal amounts of each library were sequenced in parallel with unrelated samples of non-clonal DNA to add sequence diversity and facilitate cluster generation. High throughput sequencing was performed for 300 cycles using MiSeq Reagent Kit version 2 (Illumina Inc.), with sequence data pre-processed using MiSeq software (version 2.5), exported as fastA files and read with FastQC (bioinformatics. babraham.ac.uk/ projects/fastqc).