title: GSM8601888: endosperm, ros1-7, 7DAP, rep1; Arabidopsis thaliana; OTHER
library_construction_protocol: Flowers were emasculated and pollinations were preformed 2 days after emasculation. Seven days after pollination (DAP), seeds were removed from 3-6 siliques and placed into 120 ml of Nuclei Extraction Buffer, available as part of CyStain UV Precise P kit (Partec North America #055002) on ice. Seeds were ground manually using a sterile pestle. 800ul of companion Nuclei Staining Buffer was added to ground seeds, and mixture was filtered 1-2 times through sterile Cell Trics 30um filters and kept on ice. 3C and 6C nuclei were then sorted into 200ul of Buffer ATL from Qiagen QIAamp DNA micro kit (#56304) with BD FACSAria SORP equipped with an 100mW UV laser using a nozzle size = 100 microns, Sheath pressure = 20 psi. 20ul of Proteinase K solution was added to nuclei mixture and incubated overnight at 56C. The next day, remaining steps for QIAamp DNA micro kit were followed, using the tissue protocol. DNA was eluted in 20ul of nuclease-free water and kept at -20C until all samples were prepared. Rosette leaf tissue from 4-week-old plants was collected and ground manually in Nuclei extraction buffer using a razor blade. All following nuclei preparation steps were the same as for seeds. For leaf tissue, 2C and 4C nuclei were collected using the FACSAria conditions described above. DNA extraction steps were the same as for seed nuclei. To prepare converted libraries for whole-genome sequencing of DNA methylation, we used the EM-seq kit from NEB (#E7120S). 1ul diluted 1:100 pUC19 control DNA per 10-30 ng sample DNA was spiked in as per kit instructions.  Using a Covaris, DNA samples for the non-allelic seed and leaf were sheared in 130ul of 1x TE buffer, 6x16mm tubed using conditions: [Peak power = 175 W, Duty factor = 10% Cycles/burst = 200 Time = 180 sec]. TE buffer was then exchanged for 50 ul nuclease free water using a 1X Ampure bead clean up before continuing the EM-seq protocol. Conditions were later optimized, and for the allelic endosperm experiments DNA was sheared in 50ul of 1x TE buffer, 6x16mm tubes under conditions [Peak power = 175W Duty factor = 10% Cycles/burst = 200 Time = 80 sec]. All remaining library prep steps were then performed as written in the EM-seq manual; we used 7-8 cycles of PCR amplification to amplify libraries. Libraries were pooled at approximately equimolar using library quantity quantified from Fragment Analyzer readouts prior to a final bead clean-up of remaining adapter fragments, and sequenced on a NovaSeq S4.