2.3. RXR Chromatin Immunoprecipitation (ChIP) with High Throughput DNA Sequencing (Seq)

RXR ChIP-Seq was conducted using an approach analogous to the one previously employed for acetyl-histone ChIP-Seq [27, 34]. Briefly, frozen liver was minced, crosslinked in 1% formaldehyde, homogenized, and suspended in nuclear lysis buffer in the presence of protease inhibitors, then centrifuged to recover chromatin, which was sheared in a Bioruptor Sonicator (Diagenode, Denville, NJ) to generate uniform 100-500 base pair (bp) fragments. Equal quantities of chromatin (based on protein content) were immunoprecipitated using a ChIP-grade anti-RXRα/β/γ antibody (sc-774; Santa Cruz Biotechnology, Inc., Beverly, MA) previously validated and used in RXR ChIP-Seq studies of liver tissue [35, 36]. The studies reported here adhered to the guidelines and practices recommended for analyses and quality control of ChIP-Seq data by the Encyclopedia of DNA elements (i.e., ENCODE and modENCODE) consortia guidelines [37]. These include recommendations to validate the specificity of the ChIP target transcription factor antibody by immunoblot (Supplementary Figure S1) and to assess each replicate of immunoprecipitated chromatin for quality metrics including NSC, RSC, Qtag score, and Irreproducible Discovery Rate (IDR) data. As described in Supplementary Materials, assessment of those metrics here met ENCODE guidelines' criteria (as described in detail in the Supporting Information). Of note, the replicate livers studied here came from separate animals as opposed to the independent cell cultures, embryo pools, or tissue sampling that account for most substrates of the ENCODE experiments. Based on that, we anticipated the possibility of increased variability when considering experimental design, which prompted us to use liver samples from each of 5 mouse replicates per group, retain all replicates for these analyses, and apply the additional stringencies to data analysis described below.

Immunoprecipitated DNAs and corresponding input samples were submitted to the WU GTAC for blunt ending, adaptor ligation, size selection, and amplification according to established protocols and as previously described [27, 34]. Libraries were sequenced using the Illumina HiSeq-3000 as single 50 bp reads. Raw data were demultiplexed and aligned to the most recent mouse reference genome assembly (i.e., mm10) using Novoalign (Novocraft; Selangor, Malaysia). Sequence peaks were identified by comparing data from the anti-RXR antibody immunoprecipitated samples to corresponding inputs for each replicate using MACS2 [38]. Peaks were associated to genes using Peak Annotation and VISualization (PAVIS) software [39]. Significant differences in gene-associated peak sequence abundances between experimental groups were determined using DiffBind, an open source Bioconductor package that utilizes edgeR software for statistical analysis of replicated sequence count data [30, 31]. These analyses used a Benjamini and Hochberg false discovery rate (FDR) threshold of q < 0.05 [40, 41]. We also applied the following additional stringencies: (i) ≥2-fold change in RXR liver-DNA binding between experimental groups as defined by DiffBind and (ii) identification of the gene-associated RXR-bound peak by MACS in at least 3 (out of the 4-5) replicates in each experimental group with increased binding. Gene set overrepresentation analysis was conducted on genes differentially bound by RXR using the approach described for RNA-Seq data analyses.

Efforts to conduct PPARγ ChIP-Seq analyses on these liver samples were also attempted here, but those efforts were unsuccessful based on inability to identify a PPARγ antibody meeting the ENCODE guidelines when tested on mouse liver (as summarized above and described in detail in the Supporting Information; S. Kulkarni, J. Huang, and D.A. Rudnick, unpublished observations). Therefore, in order to assess the liver PPARγ-dependence of RXR liver DNA binding in the samples studied here, RXR-ChIP-Seq studies were conducted on livers from WT and liver-specific PPARγ KO mice and the result compared.