Description

These tracks provide DNA methylation levels (RRBS) for five tissues produced by pooling DNA from three 9 week old Landrace x Yorkshire crossbred pigs, and the commercially available pulmonary alveolar macrophage cell line 3D4/2 (ATCC CRL-2845TM). Available as part of the FAANG initiative.

Display Conventions and Configuration

DNA methylation

This track displays methylation levels for covered CpG sites by default. Non-CpG site methylation can be visualised as an additional subtrack. The methylation status of each CpG and non-CpG site is represented using an 11-color gradient:

0 - 5%
5 - 15%
15 - 25%
25 - 35%
35 - 45%
45 - 55%
55 - 65%
65 - 75%
75 - 85%
85 - 95%
95 - 100%

The score of this track reports the coverage (i.e. number of reads) at each site. The score has a maximum value of 1000, with all sites covered by more than 1000 reads having a score of 1000. All sites with a minimum of 10 reads are reported. The CpG non-CpG bigBed files contain two extra columns. The first represents the coverage at each site, while the second represents the percentage of reads reporting methylation at the site.

Methods

DNA methylation was assessed from frozen tissues and a commercially available pulmonary alveolar macrophage cell line via reduced representation bisulfite sequencing (RRBS). The Landrace x Yorkshire crossbred pigs were 9 weeks old at the time of euthanasia, and all samples were stored at -80oC until processing. Pulmonary alveolar macrophage cell line 3D4/2 (ATCC CRL-2845TM) was cultured in T75 cell culture flasks (SPL Life Sciences) using an RPMI 1640 culture medium (ATCC modification, Life Technologies) supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin (Life Technologies) and 0.1 mM 2-mercaptoethanol.

DNA Isolation

Genomic DNA was extracted from frozen tissues and 3 x 107 cultured cells. Briefly, tissues were incubated in 400 µl of tissue lysis buffer (0.1 M Tris-HCl, 200 mM NaCl, 5 mM EDTA, and 0.2% SDS) with 250 µg/ml proteinase K at 55oC for 6 hours, followed by phenol extraction. Cell pellets were incubated in 400 µl of lysis buffer (10 mM Tris-HCl, 100 mM EDTA, and 0.5% SDS) at 37oC for 6 hours, followed by phenol extraction. Isolated DNA was incubated with 20 µg/ml DNase-free RNase and purified using a PowerClean DNA Clean-Up Kit (MO BIO) according to the manufacturer's protocol.

RRBS Library Preparation

RRBS library preparation was performed by BGI-Shenzhen. High-quality genomic DNA (100 ng) was restriction digested using the methyl-insensitive restriction enzyme Mspl at 37oC for 16 hours, which cuts the DNA at CCGG sites. The fragments were blunt-ended and phosphorylated, and a single A nucleotide was added to the 3' ends of the fragments in preparation for ligation to a methylated adapter with a single-base T overhang. The ligation products were purified and size-selected (40 - 220 bp) using agarose gel electrophoresis. Size-selected DNA was bisulfite-treated with the ZYMO EZ DNA Methylation-GoldTM KIT (ZYMO). The treated DNA was PCR-amplified to enrich for fragments with adapters on both ends. The final libraries were analysed by an Agilent 2100 Bioanalyzer (Agilent Technologies) and quantified by real-time PCR.

Illumina Sequencing

RRBS Illumina sequencing was performed on an Illumina HiSeq2000. The libraries were sequenced to a total read length of 49 bp from both ends (paired-read sequencing, RNA-seq) of the molecules.

RRBS Data Analysis

Raw sequencing data were processed by an Illumina base-calling pipeline. An in silico converted swine genome was produced using the BS-seeker2 v.2.0.8 bs_seeker2-build.py script (Guo et al. 2013). Clean reads were aligned to the in silico converted swine genome with BS-seeker2 v.2.0.8 using Bowtie2 v.2.1.0 (Langmead and Salzberg 2012) in local alignment mode and allowing no more than 4 mismatches/read. Methylation status was determined using the bs_seeker2-call_methylation.py script using only uniquely aligned reads, and all sites covered by a minimum of 10 reads were reported.

Credits

These data were generated and analysed by the Chankyu Park Lab at the Department of Animal Biotechnology at Konkuk University, Korea. Track hubs maintained by Kyle Schachtschneider. Please direct all questions to chankyu@konkuk.ac.kr or kschach2@illinois.edu.

References

Choi M, Lee J, Le MT, Nguyen DT, Park S, Soundrarajan N, Schachtschneider KM, Kim J, Park J-K, Kim J-H, and Park C. Genome-wide analysis of DNA methylation in pigs using reduced representation bisulfite sequencing. DNA Research 22(5):343-355

Data Availability

Raw reads are available for download from the European Nucleotide Archive database (PRJEB9561).