Mapping regulatory factors by immunoprecipitation from native chromatin

Abstract

Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin (ORGANIC) is a high-resolution method that can be used to quantitatively map protein-DNA interactions with high specificity and sensitivity. This method uses micrococcal nuclease (MNase) digestion of chromatin and low-salt solubilization to preserve protein-DNA complexes, followed by immunoprecipitation and paired-end sequencing for genome-wide mapping of binding sites. In this unit, we describe methods for isolation of nuclei and MNase digestion of unfixed chromatin, immunoprecipitation of protein-DNA complexes, and high-throughput sequencing to map sites of bound factors.

Keywords: MNase-seq; ORGANIC; chromatin; transcription; transcription factor.

Figure 1. Overview of ORGANIC protocol and MNase digestion ladder

(A) ORGANIC profiling workflow. (B) Visualization of nucleosomal ladder and sub-nucleosomal fragments from Drosophila nuclei treated with increasing amounts of MNase. Digestion with 200 units produced an ideal ladder, with >75% mono-nucleosomal fragments, and a clear smear of sub-nucleosomal DNA.

Figure 2. Size selection of ORGANIC IP DNA Fragments

(A) Example paired-end sequencing fragment length distributions from libraries made from non-size-selected and size-selected ORGANIC IP DNAs. Unbound refers to fraction not bound to SPRI beads. (B) Determination of optimal SPRI bead:DNA ratio by testing various bead:DNA ratios on 10 bp ladder and visualization of bead-bound (Bound) and supernatant (Unbound) fractions on 10% acrylamide gel. “M” indicates marker lanes. (C) Enrichment of different size classes in the SPRI bead-bound (Bound) and supernatant (Unbound) fractions visualized on a 6% acrylamide gel.

Figure 3. Anticipated Results from ORGANIC profiling

(A) Robust ORGANIC profiling across a range of input cell numbers and immunoprecipitation epitopes in yeast. Example ~50 kb window showing comparison of ORGANIC profiles of the ATPase Mot1 generated using different input cell numbers and epitopes: 250 mL starting culture with FLAG-tagged Mot1 (black track); 25, 50, and 100 mL starting cultures with FRB-GFP-tagged Mot1 and immunoprecipitated with FRB antibody (blue tracks); and 25, 50, and 100 mL starting cultures with FRB-GFP-tagged Mot1 and immunoprecipitated with GFP antibody (green tracks). (B) High resolution mapping of Trl with ORGANIC in Drosophila S2 cells. An example Trl-bound site revealed by X-ChIP-seq (black track) is resolved to a ~40bp segment by ORGANIC (brown track).