title: GSM4435275: MeDIP-seq WT Tumor rep 1; Mus musculus; MeDIP-Seq
library_construction_protocol: Tumor cells (YFPbright/lineageneg cells), pre-cancerous epidermis of interfollicular epidermis (YFPpos/CD49fhigh/CD34neg cells), and bulge hair follicle stem cells (YFPbright/CD49fhigh/CD34pos cells) were FACS-sorted using a BD FACSAria Fusion flow cytometer (BD Biosciences). Approximately 3-20x10^4 cells were sorted and lysed in 1 ml of TRIzol for RNA and DNA isolation. After adding 200 µl chloroform, samples were vortexed for 30 second and then centrifuged at 12,000 g to separate the RNA-containing supernatant from the organic phase. RNA was precipitated with 1´ volume of isopropanol, washed twice with 70% ethanol, and then used for library preparation. The interphase of the TRIzol solution (after removal of the supernatant) was precipitated adding 1´ volume of isopropanol, centrifuged for 1 h at 4°C at 13,000 g, washed twice with ethanol, and digested overnight at 55°C with proteinase K (10 mg/ml) in TE 1´ buffer. The following day, digested material was incubated 1 h at 37°C with RNase A and purified using a conventional phenol/chloroform separation. The DNA pellet was quantified, and DNA was used for library preparation for MeDIP-seq and hMeDIP-seq experiments. Purified genomic DNA (250 ng) from tumor cells was sonicated to obtain fragments of 300-700 bp. Adaptors from the NEBNext Ultra DNA Library Prep Kit for Illumina were added to the fragmented DNA. DNA was denatured for 10 min at 99°C and cooled to avoid re-annealing. Fragmented DNA was incubated overnight with 1 mg of antibodies (5-methylcytosine, Abcam cat. # ab10805; 5-hydroxymethylcytosine, Active Motif, cat. # 39769) previously cross-linked with 15 ml of Dynabeads Protein A (Life Technologies). Immunocomplexes were recovered using 8 ml for 2 h. The following morning, DNA was washed three times for 10 min each, and purified DNA was extracted using QIAquick MinElute (Qiagen). Amplified libraries were prepared using NEBNext Ultra DNA Library Prep Kit for Illumina (E7370L) following the manufacturer's instructions.
attributes.tag: GEO Accession
attributes.value: GSM4435275