‘Institute’s Ethical Committee on Animal Experiments’ at the National Institute of Nutrition, Hyderabad, India. Genomic DNA was isolated from liver of two adult Wistar rats using protocol from Qiagen DNeasy Blood & Tissue Kit (Qiagen, Valencia, USA) and 20 mg/mL RNase was used to degrade the RNA present in the sample. DNA integrity was verified by agarose gel electrophoresis. Quality and quantity of DNA was measured using Nano-Drop Spectrophotometer and Quant-iT PicoGreen dsDNA Reagent and Kits (Invitrogen, USA) respectively. Methyl-DNA immunoprecipitation sequencing (MeDIP- Seq) Before carrying out MeDIP, we sonicated genomic DNA to produce random fragments ranging in size from 100 to 500 bp and purified using the PCR purification kit (Qiagen). Based on the manufacturer’s recommended protocol, we then end-repaired, phosphorylated and A-tailed the fragmented DNA and ligated Illumina single read adapters to the fragments. We used ,4 mg of adaptor-ligated DNA for subsequent MeDIP enrichment. Briefly, following adaptor ligation, DNA was denatured at 95uC for 10 min. Immunoprecipitation was then carried out at 4uC for 3 hr using 10 mg of monoclonal antibody against 5-methylcytidine (Eurogentec) in a final volume of 500 ml IP buffer (10 mM sodium phosphate (pH 7.0), 140 mM NaCl, 0.05% Triton X-100). We incubated the mixture with 40 ml of Dynabeads with M-280 sheep antibody to mouse IgG (Dynal Biotech) for 2 hr at 4uC and washed it seven times with 700 ml of IP buffer. We then treated the beads with proteinase K for 4 hr at 50uC and recovered the methylated DNA by phenol-chloroform extraction followed by ethanol precipitation. PCR amplification by Illumina single read PCR primers was performed as described earlier. We performed agarose gel electrophoresis and excised bands from the gel to produce libraries with insert sizes of ,200 bp, and quantified these libraries using the Quant-iT PicoGreen dsDNA Reagent and Kits (Invitrogen). We then prepared flowcells with 14 pM DNA using the manufacture’s recommended protocol and sequenced for 36 cycles on an Illumina Genome Analyzer II. Obtained images were analyzed and base-called using GA pipeline software version 1.3 with default settings provided by Illumina. PCR and real-time PCR on MeDIP samples We carried out real time PCR reactions with 0.5 ng of input DNA and immunoprecipitated methylated DNA. For real-time PCR reactions, we used the SYBR Green PCR master mix (Kappa Biosystems) and Roche - LightCycler 480 System. For each qRT-PCR reaction (total volume of 10 ml), we used 5 ml SYBR Green PCR master mix and 2 ml primer mix (0.5 mM each). Reaction conditions were as follows: 1 cycle at 95uC for 30 seconds, 35 cycles at 95uC for 30 seconds and 1 cycle at Tm for 30 seconds. Experiments were done in triplicates. We have followed the method described by Tomazou et al to evaluate the relative enrichment of target sequences after MeDIP [9]. Briefly we normalized the Ct of the MeDIP fraction to the Ct of the input (DCt). Subsequently we normalised the DCt of each target sequence to the DCt of an unmethylated control sequence (DDCt). Finally, the enrichment was calculated as E = 2DDCt. Refer Table S4 for primer sequence RT-PCR Total RNA (1 mg) was isolated using the RNeasy RNA isolation kit (Qiagen) and reverse transcribed using Superscript III (Invitrogen) using random hexamers, according to the manufac- turer’s protocol. PCR were performed on 1 ml of complementary First High Resolution Rat Methylome DNA or a comparable amount of RNA with no reverse transcriptase, using AmpliTaq Gold DNA Polymerase (ABI Biosystems). The list of primers is attached in Table S4. Bisulfite Sequencing DNA (0.5 mg), from same rat liver sample was bisulfite converted using the EZ DNA methylation kit (ZYMO Research) according to the manufacturer’s protocol.PCR was performed using primers flanking the methylated and unmethylated regions. PCR products were cloned into pGEMH-T (Promega). Randomly clones were sequenced and then analyzed using BiQ Analyzer [12]. Refer Table S4 for primer sequence. Data Download and Analysis We downloaded the rat genome sequence and mapping information (rn4) from the University of California Santa Cruz Genome Bioinformatics Site (http://genome.ucsc.edu). The reads were mapped onto the rat genome reference sequence using the high-performance alignment software ‘maq’ version 0.7.1 (http:// maq.sf.net) and those with maq quality less than 10 were removed from further analysis. We used MACS (version 1.4.0 beta) for peak detection and analysis of immunoprecipitated single-end sequenc- ing data to find genomic regions that are enriched in a pool of specifically precipitated DNA fragments. The Browser Extensible Data (BED) files of the Human Brain MeDIP seq was downloaded from the SRA012488 [14]. These BED files were then merged and analyzed by MACS to generate peak summit coordinates. The summit files were then used for further downstream analysis. The data for the analysis of alternate splicing events was downloaded from the EBI ASTD database version 1.1 (http://www.ebi.ac.uk/astd/main.html;jsessionid = 8E5318CC1D7E9AF0E003465EE3084922).The IPI IDs of identi- fied liver proteins were searched for their gene IDs in ENSEMBLE genome browser and then in UCSC Genome Bioinformatics for gene coordinates. Of the 524 proteins, we could get 494 gene IDs and further analysis was done using these proteins. For analyzing the methylation pattern between the highly vs lowly expressed genes we downloaded microarray gene expression data for control rat liver from Gene Expression Omnibus (GSE 19830). Data analysis was done using Bioconductor package Affy, via R programming language. Average of the normalized intensities of all three replicates was converted to log base 2, and then statistically highly and lowly expressed (mean 6 standard deviation) genes were used to check the methylation pattern across their TSS in a 100 kb sliding window. The RefSeq genes, repeat element and CGI coordinates of human and rat were downloaded from UCSC Genome Bioinfor- matics. The CGIs in our study follow the three basic character- istics, a) length greater than 200 bp, b) GC content .50% and c) CpG Observed/Expected .0.6. The methylation status of the CpG Islands was determined by mapping the methylation peak summits (from MeDIP-Seq data) on the CpG islands. Islands having methylation peak summits were designated methylated islands while the rest were termed unmethylated. For describing the methylation of any event, we have used the term ‘‘methylation density’’, which in the case of all bar plots is the ratio of methylation peak summit count in the given region to the area in base pairs of that region (Figure 1, 2, 7). While in the case of line plots, methylation density refers to the ratio of methylation peak count vs number of data points (Figure 3, 4, 5). 1D SDS-PAGE and In-Gel Tryptic Digestion Briefly, 100 mg of total protein of rat liver was separated in 12% SDS-polyacrylamide gel using the Biorad SDS-PAGE setup. The PLoS ONE | www.plosone.org 9 February 2012 | Volume 7 | Issue 2 | e31621