BS-seq and OxBS-seq reads were trimmed and then mapped to the bisulfite-converted human hg19 reference genome with bowtie2 (52). The calling of unmethylated and methylated base calls was performed by Methylpy (https://github.com/yupenghe/methylpy) (12).

Bisulfite conversion efficiency for each type of cytosines was estimated with CEGX spike-in control sequences (table S3). In each sample, let RC_BS, RmC_BS, and RhmC_BS be the conversion rates (fraction of bases read as thymine in sequencing) for unmethylated Cs, methylated Cs, and hydroxymethylated Cs in the BS-seq data, respectively. Similarly, let RC_OxBS, RmC_OxBS, and RhmC_OxBS be the conversion rates for unmethylated Cs, methylated Cs, and hydroxymethylated Cs in the OxBS-seq data, respectively. Thus, we haveEmbedded Imagewhere C′, mC′, and hmC′ are the observed percentages of unmethylated Cs, methylated Cs, and hydroxymethylated Cs in a particular region estimated from the BS-seq and OxBS-seq data, and C, mC, and hmC are the adjusted levels of unmethylated Cs, methylated Cs, and hydroxymethylated Cs, respectively. Nonconversion rate adjustment was done by solving the system of equations above.

DMRs for total methylation (tmC) and true methylation (mC) were defined using Dispersion Shrinkage for Sequencing (DSS) (53) with some modification. Differentially methylated loci (DMLs) were called using a modified “callDML” function, where the difference of mean methylation level between the two samples was compared with the global methylation difference using a Wald test at each CpG site. CpG sites with FDR-corrected P value less than 0.05 were deemed as DMLs. DMRs were then called with the “callDMR” function, and we required each DMR to contain at least three DMLs, to have a minimum length of 50 bp, to have an absolute difference of methylation level greater than 0.3 relative to the global methylation difference in the two cell types in comparison, and to have a detected P value less than 0.05.

Because hmC is indirectly inferred on the basis of the difference between BS and OxBS signals, DSS and other published DMR calling methods are not applicable to hmC in our data sets. To call DMRs for hydroxymethylation (DhMR), the genome was tiled by 1-kb bins. hmCG levels in these bins were estimated and adjusted for nonconversion rate using both BS-seq and OxBS-seq data. DhMRs were then defined as bins that have a difference of hmCG levels greater than the global difference of +0.3 in the two cell types in comparison.

RNA-seq reads were trimmed to remove sequencing adapters and low-quality sequences using Cutadapt (54) in paired-end mode. The first 3 bp that form the 5′ end of read 1 were also removed. Trimmed reads were then mapped to the hg19 genome and the GENCODE annotated transcriptome (V25, coordinates lifted to hg19 with liftOver) with STAR (Spliced Transcripts Alignment to a Reference) (55). Gene expression was estimated using RSEM (RNA-Seq by Expectation Maximization) (56), and DE genes were called using edgeR (57) in exact test mode requiring FDR < 0.05 and FC > 2 (table S2).

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