GSM1904283: H3K4me3.B6.PRDM9bb; Mus musculus; ChIP-Seq
The testis tunica was removed, the tubules disassociated with tweezers and fixed in 1% formaldehyde in PBS for 5 minutes followed by glycine quenching (125mM final conc.) for 5 minutes at room temperature. Following washing steps, pellets were resuspended in 900 μl cold RIPA lysis buffer, dounced 20 times and sonicated in 300 ul aliquots in a Bioruptor Twin sonication bath at 4°C for three 10-minute periods of 30s on, 30s off at high power, then cell debris was pelleted and removed and aliquots were pooled. For each sample, 50 μl of equilibrated magnetic beads  (Invitrogen M-280 Sheep Anti-Rabbit Dynabeads) were resuspended in 100 μl PBS/BSA and added to the chromatin samples for pre-clearing for 2h at 4°C with rotation. Beads were removed, and 100 μl of pre-cleared chromatin was set aside for the input control. 5 μl rabbit polyclonal anti-H3K4me3 antibody (Abcam ab8580) was added to the remaining pre-cleared chromatin and incubated overnight at 4°C with rotation. 50 μl beads were washed and resuspended as before, then incubated with the chromatin samples for 2h at 4°C with rotation. Beads were then washed and decrosslinked at 65°C, and for input controls, 50 μl of pre-cleared chromatin was used. After descrosslinking, samples were further incubated with 80μg RNAse A at 37°C for 60 minutes and then with 80μg Proteinase K at 55°C for 90 minutes. DNA was purified using a Qiagen MinElute reaction cleanup kit.     ChIP and total chromatin DNA samples were sequenced in multiplexed paired-end Illumina libraries, yielding 51bp reads. We prepared two biological replicates plus one genomic input control each for the Infertile (PWD×B6)F1_PRDM9-PWD/B6, Reciprocal (B6×PWD)F1_PRDM9-B6/PWD and Rescue (PWD×B6)F1_PRDM9-PWD/H mice, yielding roughly 40-50 million usable read pairs per replicate. For the B6_PRDM9-B6/B6 and B6_PRDM9-H/H mice, we prepared one biological replicate each (yielding 70-80 million usable read pairs per sample) and later split read pairs into pseudoreplicates. Sequencing reads were aligned to mm10 using BWA37 (options -q 10 -t 8) followed by Stampy38 (options -t 8 --bamkeepgoodreads), and reads not mapped in a proper pair with insert size smaller than 10kb were removed. Read pairs representing likely PCR duplicates were also removed by samtools rmdup. Pairs for which neither read had a mapping quality score greater than 0 were removed. Fragment coverage was computed using in-house code and the samtools and bedtools packages. Libraries were prepared according to Illumina's instructions. The Infertile and Rescue samples were multiplexed together, as were the Reciprocal and B6 samples.
GEO Accession
GSM1904283