Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single nucleotide resolution
Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single nucleotide resolution
s_5_J1_P20_RRBS_1
J1 ES cells (129S4/SvJae) were purchased from ATCC (Cat. SCRC-1010) and cultured on a g-irradiated pMEF feeder layer at 37C and 5% CO2 in complete ES medium (DMEM 4500 mg/L glucose, 4 mM L-glutamine and 110 mg/L sodium pyruvate, 15% fetal bovine serum, 100 U of penicillin/100 ug of streptomycin in 100 mL medium, 0.1mM non-essential amino acids, 50 mM b-mercaptoethanol, 103U LIF ESGRO). Genomic DNA was prepared from ES cells at passage 14 or 20 using the Qiagen Allprep DNA/RNA mini kit. RRBS libraries from oxidised and non-oxidised DNA were prepared based on a previously published protocol (H. Gu et al., Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling. Nature protocols 6, 468 (Apr, 2011)). Briefly, 2 ug of genomic DNA were digested with MspI (Fermentas) followed by end repair and A-tailing with Klenow (Fermentas) and ligation of methylated adaptors (Illumina) with T4 DNA ligase (NEB). Adaptor-ligated MspI-digested DNA was run on a 3% agarose gel and size selected (110-380 bp), followed by purification with the Qiagen QIAquick gel purification quick and ethanol precipitation. Prior to oxidation, size-selected DNA was filtered through a mini quick spin oligo column (after four 600 uL H2O washes) to remove any last remaining buffers/salts and adjusted to a final volume of 25 uL. 5 uL of this solution were kept for generation of the non-oxidised library. The remaining was oxidised according to the general oxidation for genomic DNA. Both oxidised and non-oxidised DNA samples were bisufite-treated using the Qiagen Epitect kit, according to the manufacturers instructions for FFPE samples, except that the thermal cycle was run twice over. Final library amplification (18 cycles) was done using Pfu Turbo Cx (Agilent) and adaptor-specific primers (Illumina), after which the libraries were purified using AMPure XP beads (Agencourt).