Nextflex chip seq kit

Antibodies: The anti-Vegt (Sudou et al., 2012 (link)), anti-Foxh1 (Chiu et al., 2014 (link)), anti-Pol II (Bio-Legend
MMS-126R; Charney et al., 2017a (link)), and
anti-H3K4me1 (Abcam; Gupta et al.,
2014; Hontelez et al.,
2015) antibodies were previously tested. The rabbit polyclonal
anti-Otx1 peptide antibody was generated (GenScript USA Inc.) against the
peptide sequence GYTGTGLPFNSSDC (AA 288–301 of the Xenopus
tropicalis Otx1 protein). ChIP: ChIP on X.
tropicalis embryos was performed as previously described (Chiu et al., 2014 (link)). ChIP-qPCR
Primers: See Table S5. ChIP-qPCR: qPCR was performed using Roche
Lightcycler 480 II using the Roche SYBR green I master mix with the default
SYBR green protocol. ChIP-seq: ChIP-seq libraries were
generated using Nextflex ChIP-seq kit (Bioo Scientific), analyzed using an
Agilent Bioanalyzer 2100, quantified using KAPA qPCR and sequenced using
Illumina instruments at the UC Irvine Genomics High Throughput Facility.
Sequential ChIP: The initial steps of ChIP were performed
as previously described. After incubation with the first antibody, elution
was performed using 1x TE, pH 8.0, with 10mM DTT, 500 mM NaCl and 0.1% SDS
at 37°C for 30 minutes. After the incubation, the eluate was diluted
10x with RIPA. The diluted eluate was incubated with the second antibody and
the rest of the ChIP was performed as previously described (Geisberg and Struhl, 2004 (link)).

ChIP was performed using antibody against H3K4me3 (Millipore, #07-473) as previously described (Billings et al. 2013 (link)). To obtain sufficient material for high-throughput sequencing, four ChIP reactions from a single preparation of spermatocyte chromatin were pooled after final DNA elution. ChIP DNA was concentrated using Agencourt AMPure XP beads (Beckman Coulter) following the manufacturer’s protocol. Quantitation of ChIP and input DNA was measured using the Qubit dsDNA HS assay (Life Technologies). Typically, 7–10 ng of ChIP DNA was obtained after concentration and used for the sequencing library preparation. For all samples, an equal amount of MNase-treated input DNA was sequenced as a control. Biological replicates for each genotype were prepared independently from different litters of mice. Libraries were prepared for sequencing using Bioo Scientific’s NEXTflex ChIP-Seq Kit (protocol version V11.11) without size selection. Amplification of the libraries was done with 20 µL ligation product, 16 µL water, 12 µL NEXTflex ChIP PCR master mix, 2 µL NEXTflex ChIP primer mix, and 14–18 cycles of PCR.