GSM2813757: spt16/+ EM BS-seq; Arabidopsis thaliana; Bisulfite-Seq
Arabidopsis flower buds were emasculated at flower stage 12-13 using fine forceps and pollinated with Ler pollen 48 hours later. Eight to ten days after pollination (DAP) developing F1 seeds (torpedo to bending cotyledon stage) were immersed in dissection solution (filter-sterilized 0.3 M sorbitol and 5 mM pH 5.7 MES) on sticky tape and dissected by hand under a stereo-microscope using fine forceps (Fine Science Tools, Inox Dumont #5) and insect mounting pins. The seed coat was discarded, and debris removed by washing collected embryos or endosperm five to six times with dissection solution under the microscope.  For ssrp1-3 and spt16-3 mutant normal and delayed sibling seed collection, seeds were divided into fractions according to embryo development. At 8 DAP, delayed seeds tended to be at the torpedo to early-linear cotyledon stages, whereas normally developing seeds were late-linear cotyledon to bending cotyledon stages.  Seeds with an intermediate developmental stage were discarded. Vegetative cell and sperm nuclei were extracted from mature pollen and fractionated by fluorescence activated cell sorting as described previously. 50 ng of genomic DNA was fragmented by sonication, end repaired and ligated to custom-synthesized methylated adapters (Eurofins MWG Operon) according to the manufacturer's instructions for gDNA library construction (Illumina).  Adaptor-ligated libraries were subjected to two successive treatments of sodium bisulfite conversion using the EpiTect Bisulfite kit (Qiagen) as outlined in the manufacturer's instructions. The bisulfite-converted library was split between two 50 ul reactions and PCR amplified using the following conditions: 2.5 U of ExTaq DNA polymerase (Takara Bio), 5 _l of 10X Extaq reaction buffer, 25 _M dNTPs, 1 _l Primer 1.1 and 1 _l multiplexed indexing primer.  PCR reactions were carried out as follows: 95¼C for 3 minutes, then 14-16 cycles of 95 ¼C 30 s, 65 ¼C 30 s and 72 ¼C 60 s. Enriched libraries were purified twice with AMPure beads (Beckman Coulter) prior to quantification with the Qubit fluorometer (Thermo Scientific) and quality assessment using the DNA Bioanalyzer high sensitivity DNA assay (Agilent)
GEO Accession
GSM2813757