Streamlined quantitative analysis of histone modification abundance at nucleosome-scale resolution with siQ-ChIP version 2.0

Abstract

We recently introduced an absolute and physical quantitative scale for chromatin immunoprecipitation followed by sequencing (ChIP-seq). The scale itself was determined directly from measurements routinely made on sequencing samples without additional reagents or spike-ins. We called this approach sans spike-in quantitative ChIP, or siQ-ChIP. Herein, we extend those results in several ways. First, we simplified the calculations defining the quantitative scale, reducing practitioner burden. Second, we reveal a normalization constraint implied by the quantitative scale and introduce a new scheme for generating ‘tracks’. The constraint requires that tracks are probability distributions so that quantified ChIP-seq is analogous to a mass distribution. Third, we introduce some whole-genome analyses that allow us, for example, to project the IP mass (immunoprecipitated mass) onto the genome to evaluate how much of any genomic interval was captured in the IP. We applied siQ-ChIP to p300/CBP inhibition and compare our results to those of others. We detail how the same data-level observations are misinterpreted in the literature when tracks are not understood as probability densities and are compared without correct quantitative scaling, and we offer new interpretations of p300/CBP inhibition outcomes.

RepEnTools: an automated repeat enrichment analysis package for ChIP-seq data reveals hUHRF1 Tandem-Tudor domain enrichment in young repeats

“…First, we normalise to the library size (the sum of mapped primary reads), effectively normalising to sequencing depth, and thereby converting the summary counts over RE types to probabilities within the specific ChIP-seq dataset. A conceptually similar approach uses probability distributions for quantitative ChIP-seq, albeit in a significantly more complex manner [ 30 ]. Next, we use the normalised counts to calculate the ChIP over input ratio and determine enrichment or depletion of specific RE groups.…”

Select EZH2 inhibitors enhance viral mimicry effects of DNMT inhibition through a mechanism involving NFAT:AP-1 signaling

“…We then used our recently developed sans spike-in quantitative chromatin immunoprecipitation sequencing (ChIP-seq) method (siQ-ChIP) ( 59 , 60 ) to quantify changes in H3K27me3 after drug treatment (fig. S4, D to G).…”

“…Immunoprecipitated fragments and saved inputs were quantified with the Qubit dsDNA High Sensitivity Assay kit (Invitrogen, Q32851), and 10 ng of purified DNA for each IP and input sample were used for library preparation with the KAPA Hyper Prep Kit (Kapa Biosystems, KR0961). TAZ and combo treatments required two IPs per biological replicate to attain enough material for library preparation, and this doubling has been accounted for in the parameters for siQ-ChIP ( 59 , 60 ) for these samples. Library preparation including fragment end-repair, A-tail extension, and adapter ligation was conducted per the manufacturer’s instructions (KAPA Biosystems).…”