GSM1977638: xl_st10.5_MethylC-seq

GSM1977638: xl_st10.5_MethylC-seq

Embryos were homogenized in 3 volumes STOP buffer (15 mM EDTA, 10 mM Tris-HCl pH7.5, 1% SDS, 0.5 mg/mL proteinase K) after which two phenol:chloroform:isoamylalcohol (PCI, 25:24:1) extractions were performed. DNA was then precipitated in 1/5 volume NH4AC 4M and 3 volumes EtOH and dissolved in 100 uL, treated with RNase and purified by a PCI extraction. The precipitation was done with EtOH and the DNA was resuspended in 50 uL H2O.
MethylC-seq library generation was performed as described previously (Ulrich et al, 2015; Nature Protocols). Briefly, the genomic DNA was sonicated to an average size of 200 bp using a Covaris sonicator. Sonicated DNA was then purified and end-repaired followed by the ligation of methylated Illumina TruSeq sequencing adapters. Library amplification was performed with KAPA HiFi HotStart Uracil+ DNA polymerase (Kapa Biosystems, Woburn, MA), using 6 cycles of amplification.

MethylC-seq libraries were sequenced in single-end mode on the Illumina HiSeq 1500 platform. The sequenced reads in FASTQ format were mapped to the in silico bisulfite-converted reference genome (Xenopus laevis: J-Strain v9.1) using the Bowtie alignment algorithm with the following parameters: -e 120 -l 20 -n 0 as described before (Lister et al, 2009; Nature).
Genome_build: Xenopus laevis genome (J-Strain v9.1)
Supplementary_files_format_and_content: .txt file reports DNA methylation calls (CG context)
col1: chromosome name, col2: sequence start, col3: sequence end, col4: C calls, col5: coverage