Quantitative multiplexed ChIP-Seq (MINUTE-ChIP) Calibration Experiment

In MINUTE-ChIP, native chromatin is fragmented using Micrococcal nuclease, and subsequently blunted and ligated to double-stranded DNA adaptors that include a T7 RNA polymerase promoter and a sample barcode sequence. Finally, samples are combined and subsequent ChIP reactions are performed with the pooled samples. ChIP material is prepared into an Illumina-compatible library using linear amplification by virtue of T7 RNA polymerase, reverse transcription and a low-cycle library PCR amplification. Native MINUTE-ChIP is based on Mint-ChIP, developed by the Bernstein lab (van Galen et al., 2016; PMID: 26687680). We have introduce unique molecule (UMI) counting and paired-end mapping of the chromatin fragments to this method, which we then termed MINUTE-ChIP for multiplexed indexed unique molecule T7 amplification end-to-end sequencing. Here, we generate a standard curve for H3K27me3 and demonstrate that MINUTE-ChIP has a large linear dynamic range, thus MINUTE-ChIP quantitation is proportional to real quantities. Overall design: We treated mESC either with EZH2 inhibitor EPZ-6438 to reduce H3K27me3 below detectable levels, or with an inhibitor to demethylases JMJD3/UTX, GSK-J4, to increase H3K27me3 above physiologic levels. Mixing cells from these two treatments, H3K27me3 low (L) and H3K27me3 (H), in defined ratios (100:0, 95:5, 75:25, 50:50, 25:75 5:95, 0:100 L:H), we created an artificial gradient of H3K27me3. Two replicates were prepared from each mixing ratio using two different barcodes and pooled for MINUTE-ChIP. From this pool, ChIP was performed in parallel using H3K27me3, H3K27me2, H3K27me1 and H3 antibodies. After demultiplexing reads by barcode and UMI-deduplication, reads were mapped to the mm9 genome. Total mapped read counts per barcode were normalized to the respective input read counts, essentially correcting for uneven barcode representation in the input. The resulting Input Normalized Mapped Read Count (INRC) is a quantitative, proportional measure of the abundance of ChIP epitope, e.g. H3K27me3, for each barcode.