PHG86 CENH3-ChIP-seq
We collected seedling leaves from 30 to 70 seedlings per sample when the seedlings were ~12 cm in height. We obtained all the B73-related inbreds stocks as well as one of our B73 stocks (B73 “distant kin” in Table 2) from GRIN National Genetic Resources Program (http://www.ars-grin.gov). The other B73 samples (B73 “reference” and “selfed siblings” in Table 2) were from a B73 lineage that had been separated from the GRIN B73 for at least eight generations.  We carried out native ChIP on these samples using anti-CENH3 (maize) antibodies [Nagaki et al (2003), Chromatin immunoprecipitation reveals that the 180-bp satellite repeat is the key functional DNA element of Arabidopsis thaliana centromeres, Genetics; and Zhong et al (2002) Centromeric retroelements and satellites interact with maize kinetochore protein CENH3, Plant Cell]. Briefly, we extracted chromatin from finely ground, frozen tissue, digested with Micrococcal nuclease (MNase) to reduce the chromatin to mainly mononucleosomes, before immunoprecipitating to select for CENH3 nucleosomes. We used Protein A sepharose to purify the antibody/nucleosome/DNA complexes. Immediately after pre-cleaning and before incubating with antibodies, a portion of the B73 reference sample was removed and set aside as the input sample (B73 reference input).    For all samples, we started with 100-300 ng of double-stranded, fragmented DNA and end-repaired and 5'-phosphorylated using the End Repair Module (New England Biolabs #E6050L), A-tailed with Klenow exo- (New England Biolabs #M0212S), ligated to adapters (8.3 nM) with a Quick Ligase Kit (New England Biolabs, #M2200S, and PCR amplified using Phusion 2X HF master mix (New England Biolabs #M0531S) in a 50-ul total volume containing 200 nM of each primer. Thermocycler conditions were as follows: An initial denaturation at 98°C for 30 seconds; no more than 12 cycles of 98°C for 30 seconds, 65°C for 30 seconds, and 72°C for 30 seconds; and a final extension at 72°C for 5 minutes. We purified DNA between each enzymatic treatment using Agencourt AMPure XP at a ratio of 1.8:1, except after PCR, where the ratio was 0.9:1. Prior to sequencing, we selected amplicons corresponding to ~150-bp inserts by gel electrophoresis on a 2.5% agarose gel and purified using a QIAquick gel extraction kit (Qiagen #28704). We used indexed PCR primers from the ScriptSeq Index PCR primers (Epicentre, #RSBC10948 and #SSIP1202) at a concentration of 200 nM. We used a Y adapter of the following two oligos at a concentration of 8.3 nM each: /5Phos/GATCGGAAGAGCACACGTCT and ACACTCTTTCCCTACACGACGCTCTTCCGATCT.
PHG86 CENH3-ChIP-seq