Protein g agarose salmon sperm dna

ChIP assay was performed as previously described21 (link). In brief, nuclear fractions were extracted as chromatin from sonicated cells. After preclearance with protein G agarose/salmon sperm DNA (Millipore, Billerica, MA), chromatin fractions were incubated with anti-mouse/human KAT13D/CLOCK antibody ChIP grade (rabbit polyclonal IgG) (ab3517, Abcam, Cambridge, MA), anti-mouse SLC22A3 (OCT3) Ab (ab191446, Abcam, Cambridge, MA) or control purified rabbit IgG (no. 026102, Invitrogen, Inc., Carlsbad, CA). The “input” samples were DNA extracts prepared from untreated chromatin, and DNA was extracted from immunoprecipitated chromatin. Equivalent masses of immunoprecipitated and input DNA were analyzed by real-time PCR using primers and a TaqMan probe for the promoter region of OCT3 (Supplementary Fig. S7), mPer2-E2, and mPer2-E5 (ref. 22 (link)) as follows:
OCT3Sense (5′-GGAGCTGGAGGAATGTGATGAC-3′),
Antisense (5′-CTCCAGGAACAGAGTTTCATACCT-3′),
TaqMan probe (5′-FAM-CTGCACCTGCACCTGC-MGB-3′).
mPer2-E2,
Sense (5′-CCACCAATTGATGAGCGTAGC-3′),
Antisense (5′-CGTCGCCCTCCGCTG-3′),
TaqMan probe (5′-FAM-TCACGTTTTCCACTATGTG-MGB-3′).
mPer2-E5,
Sense (5′-TCCTGCCACATTGAGATTTGG-3′),
Antisense (5′-GTGATTGCCCCACACTCACA-3′),
TaqMan probe (5′-AAGAGATGGCACGTTAGT-MGB-3′).
Data are presented as the ratio of the cycling threshold value of immunoprecipitated DNA to that of input DNA.

~150 pairs of 3-day-old female ovaries obtained from young parents (F7) were dissected for every IP experiment. ChIP was performed according to the published procedure [67 (link)]. 2.5 μg and 50 ng of chromatin were taken for each ChIP and input probe, respectively. Protein G Agarose/Salmon sperm DNA (Millipore) was used without preincubation with chromatin. Chromatin was immunoprecipitated with rat Rhi antiserum [68 (link)]. Quantitative PCR was conducted with Lightcycler96 (Roche). Percent input was calculated using the formula: % input = (2^(Ct input–Ct IP)) × F_d × 100, where % input is the ChIP efficiency expressed in percent when compared with total DNA; Ct IP and Ct input are threshold cycles for ChIP and input samples, respectively; F_d is the dilution factor. SEM of triplicate PCR measurements for two biological replicates was calculated.

The coimunoprecipitation assay were carried out as previously described23 (link). Briefly, RAW264.7 cells were plated in 10-cm dish and treated with 30 ng/ml RANKL for 48 hours and then cross-linked with 1% formaldehyde for 10 min at room temperature before harvest. After sonicated, the sample was pre-cleared with Protein G Agarose/Salmon Sperm DNA (Millipore) at 4 °C for 1 h with rotation, and 1% of the pre-cleared chromatin was set aside as the input control. Immunoprecipitation was carried out with 5 μg of antibody overnight at 4 °C against NFATc1 or Rabbit IgG (Santa Cruz Biotechnology). Immune complexes were pulled down and subjected to quantitative real-time PCR. Results were normalized to the input control. Primer for amplifing the NFATc1 binding regions of the mouse cathepsin K promoter are 5′-CCCCCAAAGTCAGTCAGATG-3 and 5′-GGTAAGGATTGCGGAAGTCA-3′.