Illumina NovaSeq 6000 paired end sequencing; Distinct chromatin signatures in plant male germline cells - RNA-seq
Distinct chromatin signatures in plant male germline cells - RNA-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. 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.  Library were purified with VATHS DNA purification beads with 1.2X ratio For sperm and vegetative nuclei mRNA-seq analysis, around 1000 nuclei per sample were freshly sorted into 15 ul lysis buffer ( 5% PEG8000, 0.1% Triton X-100, 0.5Unit/ul RNase inhibitor, 0.5mM each dNTPs, OligodT30VN) and immediately spun down and frozen at -80C for storage . cDNA synthesis was performed using Smart-seq2 with Maxima H-minus RT enzyme and KAPA HiFi master mix. cDNA quality control was assessed using S1 Kit on Qseq 100 and libraries was generated by barcoded Tn5 transposome.