title: GSM4217862: SMXL5_GFP+_S11; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: 2nd internode of Arabidopsis inflorescence stem were collected in a petri dish on ice. 2ml cold isolation buffer (20mM Tris (pH=7.5), 40mM NaCl, 90mM KCl, 2mM EDTA (pH=8.0), 0.5mM EGTA, 0.05% Triton X-100, 0.5mM Spermidine, 0.2mM Spermine, 15mM 2-Mercaptoethanol, 0.5mM PMSF, cOmplete Protease Inhibitor Cocktail (Roche, #11697498001)) supplemented with 10µl RiboLock RNase inhibitor (40U/µL) (ThermoFisher, #EO0381) was added to 2g of stem. Samples were chopped manually on ice for 10 min. Nuclei were collected by centrifuge (1000g, 10min, 4°C) and gently washed by cold resuspension buffer (same formula as isolation buffer but without Triton X-100) for two times. 300µl of resuspension buffer supplemented with 10µg/ml Hoechst 33342 at final concentration and 5µl RiboLock RNase inhibitor were added to the centrifuged nuclei. 15,000 nuclei were sorted into TRIzol solution as a replicate sample by BD FACSAriaTM IIIu cell sorter using a 70 µm sort nozzle according to GFP signal level, except for LTP1_GFP+_S21 which was 10,800 nuclei. RNA was extracted into 15 µl water according to manufacture's protocol. 1 µl was used for mRNA-seq library construction by Smart-seq2 protocol (Picelli et al., Nature Methods, 2013; Hofmann et al., Plant Reprod., 2019). NEBNext Ultra II gDNA prep kit with the NEBNext Multiplex Oligos for Illumina was used for library preparation
attributes.tag: GEO Accession
attributes.value: GSM4217862