protein lane was excised from the gel and chopped into 5 equal fractions. These fractions were digested with trypsin (promega V511A) as mentioned earlier [48]. Nano-RP-LC MS/MS analysis- Nanoflow LC MS was performed by coupling a split-free nano LC system (Proxeon nano-LC) with the LTQ Orbitrap mass spectrometer (Thermo Electron, Bremen, Germany). A 3 cm pre- column (100 mm i.d) packed with 5 mm Synergi C18 100 A˚ reverse- phased material was connected via a micro-tee fitted with an electrode for voltage application. This was connected to a 10 cm fused silica microcapillary analytical column (100 mm i.d) with a homemade laser pulled spray tip packed with 5 mm Synergi C18 100 A˚ reversed phase resin. Each fraction was loaded using a proxeon auto sampler and injected onto a sample storage loop. After equilibrating the columns with 30 ul buffer A at a flow rate of 6 ul/min and 10 ul buffer A at a flow rate of 0.8 ul/min respectively, the sample stored in the loop was loaded onto the trap column for desalting and then onto the analytical column for reverse-phased separation of separation of peptides. A stepwise gradient of the organic phase (Buffer B- 100% and 0.1% formic acid) with a constant flow rate of 300 nl/min was run for a total of 140 min. The composition of the gradient is as follows- 1% Buffer B for 20 min, 45% for 110 min and 100% for 2 min extended to 100% buffer B for 8 min. Nitrogen gas used as sheath (75psi) and auxiliary gas(10 units) gas with the heated capillary at 200uC. CID experiments employed helium with 35% collision energy. The resolution was set to 60000 at positive polarity. The LTQ Orbitrap mass spectrometer was operated in a data dependent MS/MS mode consisting of a full scan at mass range 350– 2000 m/z at FTMS mode followed by four data-dependent scans performed in linear ion trap in which the four topmost intense ions was subjected to MS/MS. Dynamic mass exclusion was enabled with a repeat count of once every 30 seconds for a list size of 500. Protein Identification and Data Analysis The .raw spectral files containing MS and MS/MS data were submitted to Proteome Discoverer 6.0 (Thermo Scientific, San Jose, CA) and searched using Sequest algorithm in IPI rat database (IPI.rat.v3.67.). The search was performed against IPI database V3.74 with specified precursor ion mass tolerance of 10 ppm and fragment ion mass tolerance of 0.8 Dalton with 2 missed tryptic cleavages. Oxidation of methionine was set as dynamic modification while carbamidomethylation of cysteine was set as static modification. To eliminate false discovery, the spectra were searched against decoy database 1% targeted and 5% relaxed FDR. The results of all five fractions were combined to give a multi-consensus report. Online Data Submission The MeDIP-Seq data from this study have been submitted to the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih. gov/geo/) under accession no. GSE31571. Proteome data and methylation tracks can be accessed from the following links respectively URL: http://genome.igib.res.in/epigenome/medip/ rat_proteome.rar. and http://genome.igib.res.in/SSG/SSG_ MEDIP_rat_liver_control_methylation_tracks.tar.gz. Supporting Information Figure S1 MACS model for MeDIP-Seq data. The reads generated from the MeDIP sequencing was processed through MACS (Model-based Analysis for ChIP-Seq) software version 1.4.0 beta for the generation of MACS model. The fragment size First High Resolution Rat Methylome was 200 bp and the distance d between the forward and the reverse tags is 38. (TIF) Figure S2 Saturation curve. The curve shows saturation at the end on plotting the percentage of peak covered by sampling (y- axis) against the percentage of raw reads take during data reduction approach (x-axis). (TIF) Figure S3 Concordance between replicates. Read cover- age at each base pair was calculated separately for both the replicates and then Pearson’s correlation was calculated using R programming and statistical language. (TIF) Figure S4 Bisulfite validation of MeDIP Seq data. Two regions (SSG06 and SSG08) showing high methylation and one region showing no methylation (SSG04) but with a number of CpGs were sequenced after bisulfite conversion and PCR amplification. (TIF) Figure S5 Methylation density in different bins. Methyl- ation density within genomic features along with all the repeat class (DNA, RNA, LTR, LC, LINE, SINE, SATELLITE, SIMPLE REPEAT, OTHER REPEATS, and UNKNOWN REPEATS) and rRNA (a component of RNA class) was calculated. Genomic features include RefSeq exons, introns, identified liver expressed gene exons and introns. (TIF) Figure S6 CpG methylation distribution around TSS in Human, mice and rats. Average methylation density around Transcription Start Site (TSS) of 3 different species. Distribution of peak summit count in 100 bp sliding window, 5 kb upstream and downstream from the start site was calculated for all RefSeq genes of A – Human and B - Mouse. Smoothing of peaks was done by taking moving average of 5. (TIF) Figure S7 CpG methylation distribution at exon bound- aries in Human, mice and rats. Methylation density around Exon/Intron junction of 3 different species. Distribution of peak summit count in 10 bp sliding window, 200 bp upstream and downstream from the start site was calculated for all exons of A- Human and B– Mouse. Smoothing of peaks was done by taking moving average of 5. (TIF) Figure S8 Methylation density at the start site of non coding 2nd exons. Distribution of peak summit count in 10 bp sliding window, 200 bp upstream and downstream from the start site was calculated for all 2nd exons which were non coding. Smoothing of peaks was done by taking moving average of 5. (TIF) Figure S9 Methylation distribution in alternate splice events. CpG methylation distribution in two different alternate splice events. A: Distribution of peak summit count in 10 bp sliding window, 200 bp upstream and downstream from the start site of all RefSeq exons and cassette exons. Smoothing of peaks was done by taking moving average of 5. B: Distribution of peak summit count in 10 bp sliding window, 200 bp upstream and downstream from the end site of RefSeq exons and introns of Intron Retention class. Smoothing of peaks was done by taking moving average of 5. (TIF) PLoS ONE | www.plosone.org 10 February 2012 | Volume 7 | Issue 2 | e31621