Animals and tissues
Frozen mouse tissues were collected from healthy mice under the Mayo Clinic IRCUC A00001233–16. The tissues were excised and divided. One piece from each pair was flash-frozen in liquid nitrogen and stored at − 80 °C and the other was fixed in 20-fold excess volume of 10% neutral phosphate-buffered formalin (Leica Biosystems, Winnipeg, MB) for 48 h. Fixed tissue was dehydrated and embedded in paraffin using an automated tissue processor. FFPE blocks were stored at RT for at least 2 years. These samples were used to establish and optimize Chrom-EX PE protocol.
Development of Chrom-EX PE, chromatin isolation from FFPE tissues
Two 20-μm sections from the FFPE blocks derived from mouse tissues were deparaffinized with xylene substitute and were rehydrated by progressively increasing percentage of water (95/5, 70/30, 50/50, 20/80). During the progressive dehydration with different ethanol concentrations, the tissues were resuspended in 0.5 ml of chromatin stabilization buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, 0.1% Sodium deoxycholate, 20% EtOH, proteinase inhibitor cocktails) and incubated in different temperatures ranging 25 °C to 75 °C overnight. As a control, the tissues were processed without overnight incubation. After the centrifugation (21,130 x g for 5 min), the supernatant was removed. And 0.25 ml of cell lysis buffer (10 mM Tris HCl, pH 7.5, 10 mM NaCl, 0.5% IGEPAL) was added and incubated on ice for 10 min. The lysates were washed with MNase digestion buffer (20 mM Tris-HCl, pH 7.5, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2) and were incubated in the fresh 150 μL MNase digestion buffer containing proteinase inhibitor cocktails in the presence of 10 gel units of MNase (NEB, Cat.# M0247S) at 37 °C for 20 min with continuous mixing in thermal mixer. After adding the same volume of sonication buffer (100 mM Tris-HCl, pH 8.1, 20 mM EDTA, 200 mM NaCl, 2% Triton X-100, 0.2% sodium deoxycholate), the lysates were sonicated for 15 min (30 s on / 30 s off) using Bioruptor Twin (UCD-400) (Diagenode, Inc., Denville, NJ) and centrifuged at 21,130 x g for 10 min. The supernatant was transferred in a new tube. The pellet was resuspended in 100 ul of 1X FFPE Stop buffer 1 (50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 100 mM NaCl, 1% Triton X-100, 0.1% Sodium deoxycholate, 0.05% SDS) and was subjected to sonication for 15 min (30 s on / 30 s off), and centrifuged at 21,130 x g for 10 min. The supernatant was collected into the previously saved fraction. The pellet was resuspended in 100 ul of 1X FFPE Stop buffer 2 (50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 100 mM NaCl, 1% Triton X-100, 0.1% Sodium deoxycholate, 0.1% SDS) and was subjected to sonication for 15 min (30 s on / 30 s off), and centrifuged at 21,130 x g for 10 min. The supernatant was collected into the previously saved fraction and the combined soluble fraction was served as soluble chromatin. The equal volume of soluble chromatin was mixed with 2X ChIP elution buffer (20 mM Tris-HCl, pH 8.0, 20 mM EDTA, 300 mM NaCl, 10 mM DTT, 2% SDS) and incubated at 65 °C overnight. The pellet was resuspended in 1X ChIP elution buffer and incubated at 65 °C overnight. DNAs were purified using Min-Elute PCR purification kit after the treatment of RNase A and proteinase K. DNA amount was measured by the Qubit dsDNA High Sensitivity assay (Invitrogen, Q32851), and DNA size was analyzed by the Fragment Analyzer (Advanced Analytical Technologies; AATI; Ankeny, IA) using the High Sensitivity NGS Fragment Analysis Kit (Cat. #DNF-486). For the comparison of soluble chromatin yield, the ChIP-IT® FFPE Chromatin Preparation Kit (Active Motif, Cat # 53030) was utilized according to the manufacture’s instructions.
Determination of chromatin yield from mouse FFPE tissues
The tissue image in FFPE block was captured every 5 cut, and the surface area of tissue was calculated by the NIH Image J program to determine the tissue size in the cuts. Two 20-μm sections were deparaffinized with xylene substitute and were rehydrated by progressively increasing percentage of water in ethanol up to 20%. The tissues were resuspended in 0.5 ml of chromatin stabilization buffer and incubated at 65 °C overnight. The tissues were processed as described above. Purified DNA was measured by the Qubit assay. The chromatin amount from each FFPE tissue is calculated by DNA amount per tissue size.
Frozen tissues (25 mg) were homogenized for 30 s in PBS using tissue grinder (ACTGene, ACT-AG 3080). Homogenized tissues were cross-linked to final 1% formaldehyde, quenched with 125 mM glycine, and washed with TBS. The fixed homogenates were resuspended in cell lysis buffer and incubated on ice for 10 min. The lysates were washed with MNase digestion buffer and were incubated in the fresh 500 μL MNase digestion buffer containing proteinase inhibitor cocktails in the presence of 1000 gel units of MNase at 37 °C for 20 min with continuous mixing in thermal mixer. After adding the same volume of sonication buffer, the lysates were sonicated for 15 min (30 s on / 30 s off) using Bioruptor Twin and centrifuged at 21,130 x g for 10 min. The supernatants were served as chromatin input. For FFPE tissues, chromatin input was prepared from two 20-μm sections as described in the development of Chrom-EX PE. The chromatin input was incubated with anti-H3K4me3 (abcam ab8580, lot GR188707–1), anti- RNA pol II antibodies (Bethyl A300–653, lot 3), anti-H3K27Ac (CST 8173 BC) or anti-H3K27me3 (CST, 9733 s, lot 8) overnight. After adding 10–30 μL of protein G-magnetic beads, the reactions were further incubated for 3 h. The beads were extensively washed with ChIP buffer (50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 100 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate), high salt buffer (50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate), LiCl2 buffer (10 mM Tris-HCl, pH 8.0, 0.25 M LiCl2, 0.5% NP-40, 0.5% Sodium deoxycholate, 1 mM EDTA), and TE buffer. Bound chromatins were eluted and reverse-crosslinked at 65 °C overnight. DNAs were purified using Min-Elute PCR purification kit after the treatment of RNase A and proteinase K. ChIP enrichment was validated by performing qPCR in the genomic loci targeting the transcription start sites of an active or inactive gene and an intergenic region. ChIP-seq libraries were prepared using the Ovation ultralow DR Multiplex kit (NuGEN, San Carlos, CA) or the ThruPLEX® DNA-seq Kit V2 (Rubicon Genomics, Ann Arbor, MI) according to the manufacture’s instructions. The ChIP-seq libraries were sequenced to 51 base pairs from both ends on an Illumina HiSeq 2000 instrument in the Mayo Clinic Center for Individualized Medicine Medical Genomics Facility.
Real-time PCR analysis
Real-time PCR analysis was performed using SYBR Green universal PCR mixes (Bio-Rad). The following primer sequences were used in the experiments: H3K4me3- and RNA pol II-positive control locus: mGAPDH-F: 5′- CTCATCCCCGCAAAGGCGGA -3′, mGAPDH-R: 5′- TCGGACCTGGCGATGGCTCG-3′. H3K27me3-positive control locus: mT1-F: 5′- GAGACGCCGATCCGCCGAAG -3′, mT1-R: 5′- ACTCTCCACTCCCACGCGCT-3′. H3K4me3-, RNA pol II-, and H3K27me3-negative control locus: mIntergenic-F: 5′-CCTGCTGCCTTGTCTCTCTC -3′, mIntergenic-R: 5′-ATGGCCTAGGGATTCCAGCA -3′.
Mapping and analysis of ChIP-seq data
Raw sequencing reads were processed and analyzed using the HiChIP pipeline  to obtain visualization files and a list of peaks. Briefly, paired-end reads were mapped to the mouse reference genome (mm10) by BWA  with default settings, and only pairs with at least one of the ends being uniquely mapped were retained for further analysis. Duplicates were removed using the Picard tool set. (https://broadinstitute.github.io/picard/). Peaks were called using the MACS2 algorithm and SICER at FDR < 1%. Fragment size was calculated from properly mapped read pairs. Pearson correlation analysis was performed by our in-house scripts where all datasets were randomly downsized to 25 million pairs of reads. In brief, the whole genome was divided into 5-kb bins for H3K4me3 and H3K27Ac and 100-kb bins for H3K27me3 and RNA Pol II, and the number of mapped reads in each bin was calculated, in which genomic regions that have zero or missing values in all samples are excluded. The read count per bin was normalized to 25 million mapped reads (at least one end uniquely mapped and duplicates removed), or to 25 million mapped reads from non-peak regions (total - reads mapped to peak regions). The normalized counts by logarithm log2 (normalized count + 1) were used for pairwise correlation analysis with Pearson coefficient. Here, 1 is a pseudo-count to avoid an undefined error of logarithm of zero. We also profiled the average signal levels across peak center +/− 2 kb (H3K4me3 and H3K27Ac) and peak region +/− 2 kb (H3K27me3 and RNAPII) using ngs.plot . For each pair of samples, peaks from the two peak lists were first merged if they were within 1 bp (H3K4me3 and H3K27Ac) or 200 bp of each other (H3K27me3 and RNAPII). For each merged peak, a single original peak with the lowest FDR was selected as the representative and used in the plot.