S1 Fig -

(a) Western blot analysis of immunoprecipitation (IP) of BMAL1 protein in HC11 cell protein lysate using rabbit polyclonal antibody to BMAL1 (ab3350, ChIP grade, 2 μg per IP). Lane 1 = Precision Plus Protein Standard Ladder; Lane 2 = 100 μg, Lane 3 = 150 μg, Lane 4 = 200 μg, Lane 5 = 200 μg of lysate precleared and incubated with beads but no Ab (-), Lane 6 = an aliquot of supernatant pulled off of lane 5 sample before wash steps that precede elution. western blot was performed using the mouse monoclonal antibody to BMAL 1 (sc-373955 @ 1:750 primary antibody concentration) for visualization. (b) Electropherogram analysis of input DNA used for ChIP-seq shows ideal size for next generation sequencing (seq). (c) Evaluation of antibody specificity indicated no difference between mock-ChIP and BMAL1-ChIP samples in the cycle threshold values following RT-qPCR analysis of an exon region of the Magea1_2 sperm specific gene, which is not a BMAL1:CLOCK target. Whereas a 9-fold difference in enrichment was found between RT-qPCR analysis of BMAL1-ChIP and mock-ChIP for the Per1 promoter region versus the exon region of Magea1_2. A positive ChIP was defined as at least 2-fold greater than mock-IP sample. Different letters indicate significant difference at p (TIF)