title: GSM2829914: dcl2dcl4_FACS_pollen; Arabidopsis thaliana; ncRNA-Seq
library_construction_protocol: Pollen was collected in 1.5mL eppendorf tubes by vortexing open flowers in pollen extraction buffer (PEB, 10 mM CaCl2, 2 mM MES, 1 mM KCl, 1% H3BO3, 10%) for 3 min, followed by filtration through a 30um mesh (Partec/Sysmex) and centrifugation at 5,000g for 1 min. Purification of pollen expressing the GFP-miR845b sensor in dcl1-5/+ and dcl1/+,dcl2/dcl4 mutants was performed by vortexing open flowers in 1 mL of PEB and filtering through a 30um mesh before FACS. Pollen population was identified in SSC/FSC scatter plots, and GFP fluorescence was analyzed by excitation with a 488nm laser and detected with a 530/30 bandpass filter. Pollen total RNA was extracted using Trizol LS reagent (Life Technologies) by vortexing with glass beads for 5 minutes, and concentrated with Direct-zol columns (Zymo Research). Small RNAs were purified by running total RNA on acrylamide gels (15% polyacrylamide, 7M urea) and performing size selection of approximately 18 to 30-nt region using a small RNA ladder (Zymo Research). The small RNA fraction was isolated from acrylamide gel plugs by grinding with a plastic pestle in Trizol LS (Life Technologies), and concentrated using Direct-zol columns (Zymo Research). Libraries were prepared using the TruSeq Small RNA Sample Preparation Kit (Illumina) following manufacturer instructions.
identifiers.namespace: GEO
identifiers.id: GSM2829914