Illumina NovaSeq 6000 paired end sequencing; Distinct chromatin signatures in plant male germline cells - ChIP-seq
Distinct chromatin signatures in plant male germline cells - ChIP-seq
Open flower buds were freshly collected from pollen marker line into 9% sucrose solution and matured pollen grain were enriched by vortex and filtered through a 60 um nylon mesh. The sperm nuclei and vegetative nuclei were isolated by FACS using a method described previously (cite paper). Briefly, pollen grain were resuspended in ice-cold Galbraith buffer (45mM MgCl2, 30mM Sodium Citrate, 20mM MOPS, 1% Triton X-100, pH to 7.0) supplemented with 1X complete protease inhibitor cocktail (Roche) and pollen wall was then disrupted by additional vortexed at 60Hz for 30sec with glass beads (sigma). The resulting pollen wall debris were removed by filtering through a 10 um nylon mesh. The crude suspension was ready for FACS on a FACSAria III cell sorter. FACS was carried out in standard configuration using 70um nozzle. A 488nm laser was used for GFP excitation and a 561nm laser for RFP excitation.  The pollen marker line (ACT::H2B-mRFP labels vegetative nucleus, MGH3P::MGH3-eGFP labels sperm nucleus in mature Arabidopsis pollen) used in this study was kindly provided by prof. Jorg Dieter Becker (Santos et al. 2017 doi.org/10.1007/978-1-4939-7286-9_16). Plants were grown in climate controlled greenhouse under long day conditions (16hr light, 8hr dark) at 22C.  ChIPed DNA was purified using Qiagen MinElute PCR Purification Kit. Library DNA was purified using AmpureXP beads using 1:1 ratio. Antibodies used: anti-H3k27me3 (Millipore 07-449); and anti-H3K4me3 (Active Motif 39915) and  Libraries were constructed using Nextera DNA Preparation Kit.