SAMN36467864
External Id
INRAE
INSDC center name
2023-08-25T00:00:00Z
INSDC first public
2023-08-25T03:18:01.574Z
INSDC last update
SRS18311910
INSDC secondary accession
live
INSDC status
Plant
NCBI submission model
Plant.1.0
NCBI submission package
SRS18311910
SRA accession
6 weeks
age
protoplasts
cell_type
2021-05-14
collection_date
Plants from this study are in the Columbia background. They were grown in soil, in growth chambers at 20 C, in short day conditions (8 h light, from 9 am to 5 pm), at 60 % hygrometry and under a light intensity of approximatively 150 mol m-2 s-1. Bacterial infections were performed on 6 weeks-old plants by spraying. The Pseudomonas syringae pv. tomato DC3000 WT strain was grown on LB medium supplemented with 50 g/ml of rifampycin. Fresh cultures in mid-exponential phase were washed, resuspended in 10 mM MgCl2 at a final OD600=0.1, and supplemented with 0.04 % of Silwet L-77. For the three independent replicates, spraying took place at the end of the day (between 5:30 pm and 6 pm), and leaves were collected 16 h later (between 9:30 am and 10 am). During this period, plants remained uncovered. For each biological replicate, about 20-25 leaves coming from 8 independent plants were cut in thin slices with a razor blade, and digested in an enzymatic solution (1.5 % Cellulase R10, Yakult, 0.4 % Macerozyme R-10, Yakult, 400 mM Mannitol, 20 mM KCl, 20 mM MES pH = 5.7, 10 mM CaCl2, 0.1 % BSA), under vacuum at RT for 15 min, and then in growth chamber (20 C) for 50 min. The protoplast solutions were filtered through a 70 m mesh and a 40 m cell strainer, with mild centrifugation (1 min, 150 g), and resuspended each time in W5 buffer (150 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES pH = 5.7). After concentration measurements using a hemocytometer, protoplasts were recovered by centrifugation and resuspended in PBS buffer supplemented with 0.04 % of BSA at a final concentration of 1000 cells / l, just prior processing with the Chromium technology (10x Genomics). 8000 protoplasts were processed using the Next GEM Single Cell 3 library kit v3.1 (10x Genomics) following the suppliers instructions.
description
Col-0
ecotype
France
geo_loc_name
Arabidopsis thaliana
http://purl.obolibrary.org/obo/NCBITaxon_3702
organism
leaf
tissue
Plant sample from Arabidopsis thaliana
title