GSM4942825: NIH-3T3_Untreated_rep3; Mus musculus; Bisulfite-Seq
Genomic DNA was extracted from 70% confluent cell culture dishes by removing cell culture media, washing cells once with 10 mL phosphate buffered saline,  addition of 400 µL DNA lysis buffer (100mM Tris, pH 7.5, 150 mM NaCl, 0.5% SDS, 10 mM EDTA), and collection by cell scraping. The mixtures were then incubated for 30 minutes with RNAse A at 37 °C, followed by 4 hours with proteinase K at 65 °C. This was followed by phenol-chloroform extraction, precipitation in ethanol ovenight at -20 °C, washing once with 70% ethanol, and resuspension in 50 µL PCR-grade water. WGBS was performed by the Centre d'expertise et de services Génome Québec at McGill University. Genomic DNA was quantified using the Quant-iT™ PicoGreen® dsDNA Assay Kit (Life Technologies). 2x151bp paired-end libraries were generated using the NEBNext® Enzymatic Methyl-seq Kit (New England BioLabs, NEB).  Adapters were purchased from NEB. Libraries were quantified using the Kapa Illumina GA with Revised Primers-SYBR Fast Universal kit (Kapa Biosystems) and average size fragment was determined using a LabChip GX (PerkinElmer) instrument. The libraries were normalized and pooled and then denatured in 0.05N NaOH and neutralized using HT1 buffer. The pool was loaded at 225pM on an Illumina NovaSeq S4 lane using Xp protocol as per the manufacturer's recommendations. The run was performed for 2x100 cycles (paired-end mode). A phiX library was used as a control and mixed with libraries at 5% level.
GEO Accession
GSM4942825