2C-ChIP: measuring chromatin immunoprecipitation signal from defined genomic regions with deep sequencing

Abstract

Background Understanding how transcription occurs requires the integration of genome-wide and locus-specific information gleaned from robust technologies. Chromatin immunoprecipitation (ChIP) is a staple in gene expression studies, and while genome-wide methods are available, high-throughput approaches to analyze defined regions are lacking. Results Here, we present carbon copy-ChIP (2C-ChIP), a versatile, inexpensive, and high-throughput technique to quantitatively measure the abundance of DNA sequences in ChIP samples. This method combines ChIP with ligation-mediated amplification (LMA) and deep sequencing to probe large genomic regions of interest. 2C-ChIP recapitulates results from benchmark ChIP approaches. We applied 2C-ChIP to the HOXA cluster to find that a region where H3K27me3 and SUZ12 linger encodes HOXA-AS2 , a long non-coding RNA that enhances gene expression during cellular differentiation. Conclusions 2C-ChIP fills the need for a robust molecular biology tool designed to probe dedicated genomic regions in a high-throughput setting. The flexible nature of the 2C-ChIP approach allows rapid changes in experimental design at relatively low cost, making it a highly efficient method for chromatin analysis. Electronic supplementary material The online version of this article (10.1186/s12864-019-5532-5) contains supplementary material, which is available to authorized users.

A conserved HOTAIRM1-HOXA1 regulatory axis coordinates early neuronal differentiation

“…We found that depleting HOTAIRM1 slowed the rate at which cells increase confluency after RA induction (Figure 2I). Onset of this delay occurred within hours, in line with HOTAIRM1's rapid and potent induction by RA and an early role in differentiation ( [12], and results below). Cell growth was hampered until at least Day 2, and this effect was reproduced across biological replicates (Supplementary Figure S1).…”

“…When determining HOTAIRM1 RNA levels, we considered its spliced and unspliced forms, as both were found in NT2-D1, with functional implications in HOXA cluster regulation [7]. As expected, both HOTAIRM1 forms were not significantly expressed before differentiation and were potently induced by RA (Figure 2E, Supplementary Table S1) [7,12].…”

“…Given the rapid induction of HOTAIRM1 by RA in many cell models ( [7,10,12], and results below), it stands to reason that it likely functions at earlier stages of neuronal differentiation as well. We thus wondered which key genes, beyond those at the HOXA cluster, HOTAIRM1 might influence during this fundamental process.…”

LAMPS: an analysis pipeline for sequence-specific ligation-mediated amplification reads

“…When applicable (e.g., for 2C-ChIP libraries), tracks are normalized by input DNA counts and optionally corrected for sample-specific DNA density. Density correction is based on TaqMan quantification of total DNA yield following immunoprecipitation, as well as various dilution steps that occur in the preparation of pooled libraries as detailed in Wang and Cameron et al [3].…”

“…Ligation-Mediated Amplification (LMA) library preparation protocols [1,2] have become increasingly useful in targeted sequencing methodologies. Applications include Carbon Copy-Chromatin Immunoprecipitation (2C-ChIP) [3], used to study protein-DeoxyriboNucleic Acid (DNA) interactions at a defined set of loci, and Chromosome Conformation Capture Carbon Copy (5C) [4], for the targeted analysis of chromatin architecture. These assays generally produce single-read sequencing data and are often highly multiplexed.…”