title: GSM5928737: ddm1.sp.f5.bsseq; Arabidopsis thaliana; Bisulfite-Seq
library_construction_protocol: Open flowers of F4 or F5 plants (which is here denoted as F4 or F5 sperm, respectively) werecollected for pollen isolation in Galbraith buffer (45 mM MgCl2, 30 mM sodium citrate, 20 mMMOPS, 1% Triton-X-100, pH7.0) by vortexing at 2000 rpm for 3 min. Flower parts wereremoved by straining through a 40 micron filter. Pollen grains were obtained by centrifugation at2600 g for 5 minutes and broken down with glass beads (Sigma). The lysate was then transferredto a 40 micron cell strainer and the flow-through was centrifuged at 800 g for 10 min at 4°C. Thepellet was re-suspended in Galbraith buffer and stained with SYBR Green for fluorescence-activated cell sorting (FACS). Vegetative nuclei and sperm nuclei (SN) were separated andcollected based on size and fluorescence intensity. DNA was extracted from SN withChargeSwitch gDNA Micro Tissue Kit (ThermoFisher cat. no. CS11203) following manufacturer's instructions. Briefly, SN were lysed in lysis buffer with proteinase K provided bythe kit in a 55°C water bath overnight. RNA was digested with RNase A at room temperature for30 minutes, then DNA was captured with magnetic beads provided, washed, and eluted. After quantification by fluorometry, about 10-20 ng of DNA was used for bisulfite-sequencing librarypreparation with Ovation Ultralow Methyl-Seq DR Multiplex System (Nugen part no. 0336)following manufacturer's instructions. Briefly, DNA was sonicated with Bioruptor sonicator, endrepaired, ligated to adapters which contain indexes, and bisulfite converted twice with EpiTectFast DNA Bisulfite Kit (Qiagen 59802) following manufacturer's instructions. After PCRamplification, libraries were purified with Agencourt RNAClean XP Beads.