Extended Data Fig. 1: Scheme of MINUTE-ChIP workflow.
a Scheme of the MINUTE-ChIP workflow. Triplicate cell pellets for each condition were lysed and the chromatin was enzymatically fragmented to mono- and di- nucleosomes and barcoded by ligating on a dsDNA adapter. Samples were then pooled and the barcoded lysate was aliquoted to individual ChIP reactions (5% of the ChIP volume was reserved as input material and carried through protocol in a manner similar to the IPs) with magnetic beads pre-coupled with the respective antibodies. Subsequently, beads were washed and IPed DNA was eluted, proteinase K digested and purified. For constructing the final libraries, DNA from each IP was in vitro transcribed using the T7 promoter in the adapter that was ligated on in the initial step. The resulting RNA was appended with an RNA 3’ adapter (RA3) allowing for specific paired end sequencing. The RA3 in turn was used to prime the reverse transcription reaction, generating cDNA that was used as a template for the final low-cycle library PCR. At this stage, in addition to the Illumina-compatible sequences, PCR primers also carried a second barcode sequence to serve as an identifier for the IP performed. Finally, libraries are pooled and sequenced on the Illumina platform42,104. b Global genome-wide levels of H3K4me3 and H2Aub as determined by input-normalized total read counts (INRC) in naïve or primed hESC, cultured with or without EZH2 inhibitor. P values of pairwise comparisons (two-sided unpaired Student’s t test) are given. n = 3 biologically independent samples. Tracks for subsequent analysis are scaled according to the INRC values as reads per genome coverage (RPGC), with naïve serving as a reference scaled to 1x genome coverage (global average equals to 1 RPGC) c Histone H3K27me3, H3K4me3 and H2Aub levels by chromatin state. RPGC of individual replicates are shown. d Genome-wide correlation (10 kb bins, Persson-correlation coefficient) of MINUTE-ChIP replicates, including corresponding inputs. e Exemplary genome-wide comparison of 10 kb bins as scatter plot.
