ChIP-seq analysis

The seminiferous tubules from male C57BL/6 mice (P10-21) were minced and digested with accutase and 0.5 units/ml DNase II, followed by filtration through a 40 μm cell strainer (FALCON). Testicular cells were fixed in 1% formaldehyde (Thermo-Fisher) and 2 mM Disuccinimidyl glutarate (ProteoChem) in PBS for 10 min at room temperature. Crosslinked cells were lysed with LB1 (50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, and 0.25% Triton X-100) and washed with LB2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA). Chromatin lysates were prepared in LB3 (50 mM Tris-HCl pH8.0, 1% SDS, 10 mM EDTA, proteinase inhibitor cocktail (Sigma)), by sonication with Covaris S220 (Peak Incident Power, 175; Acoustic Duty Factor, 10%; Cycle Per Burst, 200; Treatment time, 600 sec; Cycle, 4).

HSF5 ChIP was performed using chromatin lysates and protein A Dyna-beads (Thermo-Fisher) coupled with 5 μg of rabbit anti-HSF5-C antibody and rabbit control IgG as described previously¹¹. After 4 h of incubation at 4 °C, beads were washed 4 times in a low salt buffer (20 mM Tris-HCl (pH 8.0), 0.1% SDS, 1% (w/v) TritonX-100, 2 mM EDTA, 150 mM NaCl), and two times with a high salt buffer (20 mM Tris-HCl (pH 8.0), 0.1% SDS, 1% (w/v) TritonX-100, 2 mM EDTA, 500 mM NaCl). Chromatin complexes were eluted from the beads by agitation in elution buffer (10 mM Tris-HCl (pH 8.0), 300 mM NaCl, 5 mM EDTA, 1% SDS) and incubated overnight at 65 °C for reverse-crosslinking. Eluates were treated with RNase A and Proteinase K, and DNA was ethanol precipitated.

ChIP-seq libraries were prepared using 20 ng of input DNA, and 4 ng of ChIP DNA with KAPA Library Preparation Kit (KAPA Biosystems) and NimbleGen SeqCap Adaptor Kit A or B (Roche) and sequenced by Illumina Hiseq 1500 to obtain single end 50 nt reads.

ChIP-seq reads were trimmed to remove adapter sequence when converting to a fastq file. The trimmed ChIP-seq reads were mapped to the UCSC mm10 genome assemblies using Bowtie2 v2.3.4.1 with default parameters. Peak calling was performed using MACS program v2.1.0⁷⁰ (https://github.com/macs3-project/MACS) with the option (-g mm -p 0.00001). To calculate the number of overlapping peaks between control IgG and HSF5-C ChIP, we used bedtools program (v2.27.1)⁷¹ (https://bedtools.readthedocs.io/en/latest/). ChIP binding regions were annotated with BED file using Cis-regulatory Element System (CEAS) v0.9.9.7 (package version 1.0.2), in which the gene annotation table was derived from UCSC mm10. Binding sites were classified using ChIPSeeker. Motif identification was performed using MEME-ChIP v5.1.1 website (http://meme-suite.org/tools/meme-chip)⁷². The motif database chosen was “JASPAR Vertebrates and UniPROBE Mouse”. BigWig files, which indicate occupancy of HSF5, were generated using deepTools (v3.1.0) and visualized with Integrative Genomics Viewer software (v.2.8.3) http://software.broadinstitute.org/software/igv/home. Aggregation plots and heatmaps for each sample against the dataset of HSF5 target genes were made using deeptools program v3.5.0. Sequencing data are available at DDBJ Sequence Read Archive under the accession DRA017059.