Oligoperfect

Total RNA was isolated from mice brains using the RNeasy Lipid mini kit
(Qiagen, Germantown, MD) and was reverse transcribed using the
RT² First Strand kit (Qiagen) from 1 μg of total RNA.
Real-time PCR analysis was performed using the StepOnePlus real-time PCR
system (Applied Biosystems, Carlsbad, CA). PCR reactions were performed in
duplicate on four independent sets of templates. Relative quantification of
gene expression for the gene targets was achieved by RT²Profiler
PCR Array Data Analysis version 3.5 (SABiosciences, Valencia, CA) using
β-actin as an internal control. Amplification was performed on a cDNA
amount equivalent to 100 ng total RNA. Oligonucleotides were designed
using Oligoperfect (Life Technologies, Carlsbad CA, sequences available upon
request). Relative quantification of gene expression was calculated by the
comparative threshold cycle (Ct) method and expressed as 2-exp
(ΔΔCt) using mouse β-actin as an internal control as
previously described.^{50 (link)}

Quantitative PCR was carried out using the Eppendorf Realplex cycler and the QuantiFast SYBR Green PCR kit (Qiagen) following the manufacturer’s protocols, except that primers were used at a final concentration of 0.5 µM to help eliminate primer dimers in later cycles. qPCR primers were designed using OligoPerfect (Life Technologies) with 40%–60%GC and 80- to 120-bp product size (primer sequences are listed in Supplemental Table S1). PCR was carried out for 40 cycles followed by melting curve analysis, and all samples were run in triplicate. Cycle number was determined using the automated threshold analysis.
For estimating the total number of PRDM9-dependent hotspots per meiosis, an equal volume of MNase-digested input DNA was analyzed alongside the ChIP DNA. Cycle threshold (Ct) values were calculated by averaging the technical replicates. Percent input was calculated by using the formula: % Input = 2^{(Ct Input−Ct ChIP)}*100.
To determine peak-to-valley ratios, the peak and valley primer pairs were assayed using five 10-fold dilutions of genomic DNA to compare amplification efficiencies. For each hotspot, after qPCR the Ct values for peaks and valleys were subtracted at each dilution and plotted against the template amount. Only peak and valley primer sets that had a delta-Ct slope ∼ 0.1 or less were used for subsequent analysis of the peak-to-valley ratio.