Chlorella H3 Autotrophic rep1
Chlorella sorokiniana
ENA first public
2024-09-12
INSDC center name
Biology Centre CAS
INSDC status
public
Submitter Id
cs_repA1
air temperature regimen
(phase 1) 30 °C, until a cell density of approximately 1×108 cells.ml-1 was reached; (phase 2) 40 °C, during 24h; (phase 3) 30 °C, during 24h; (phase 4) 30 °C, until shortly after one cell cycle
biological status
wild
collection date
2023
estimated size
58530000
genotype
UTEX1230
geographic location (country and/or sea)
Czechia
geographic location (latitude)
missing: lab stock
geographic location (longitude)
missing: lab stock
growth facility
growth chamber
isolation and growth condition
The unicellular green alga Chlorella sorokiniana CCALA 259 (equivalent to UTEX 1230) was obtained from the Culture Collection of Autotrophic Organisms at the Institute of Botany of the Czech Academy of Sciences (Trebon, Czech Republic). The cultures were grown on ½ Šetlík-Sluková (SS) medium (Hlavova et al, 2016). For routine subculture, cultures were streaked every three weeks on ½ SS medium solidified with agar (1.5 %, w/v) and grown at an incident light intensity of 100 μmol.m-2.s-1 of photosynthetically active radiation (PAR). For the experiment, 300 ml of the liquid ½ SS medium was inoculated directly from the plates and the cultures were placed in glass cylinders (inner diameter 30 mm, height 500 mm) at 30 °C and “aerated” with a mixture of air and CO2 (2%, v/v) at a flow rate of 15 l.h-1. The cylinders were illuminated from one side with dimmable fluorescent lamps (OSRAM DULUX L55W/950 Daylight, Milano, Italy), the light intensity of which was adjusted so that 500 μmol.m-2.s-1 of PAR hit the surface of the cylinders. The cultures were grown under continuous light until a cell density of approximately 1×108 cells.ml-1 was reached. They were then shifted to 40 °C and a light intensity of 700 μmol.m-2.s-1 for 24 hours to allow the cells to grow but block cell division. The cultures were then transferred to the dark at 30 °C and the cells were allowed to divide for 24 hours. These partially synchronized cultures were diluted to a cell density of approximately 1x107 cells.ml-1 either in ½ SS medium. The cultures were grown at a light intensity of 500 μmol.m-2.s-1 at 30 °C. The cultures were sampled in the early G1 phase, shortly after one cell cycle was completed.
light regimen
(routine subculture) continuous light at 100 μmol.m-2.s-1 of photosynthetically active radiation (PAR); (phase 1) continuous light at 500 μmol.m-2.s-1 of PAR, until a cell density of approximately 1×108 cells.ml-1 was reached; (phase 2) continuous light at 700 μmol.m-2.s-1 of PAR, during 24h; (phase 3) continuous darkness, during 24h; (phase 4) continuous light at 500 μmol.m-2.s-1 of PAR, until shortly after one cell cycle
mineral nutrient regimen
½ Šetlík-Sluková (SS) medium (Hlavova et al, 2016): 2020 mg/l KNO3; 11 mg/l CaCl 2⋅6H2O; 988 mg/l MgSO 4⋅7H2O; 340 mg/l KH 2PO4; 140 mg/l K 2HPO4; 3.086 mg/l H3BO3; 1.18 mg/l MnSO4⋅4H2O; 1.404 mg/l CoSO4⋅7H2O; 1.244 mg/l CuSO4⋅5H2O; 1.430 mg/l ZnSO4⋅7H2O; 1.84 mg/l (NH4)6Mo7O24⋅4H2O; 18 mg/l C10H12N2O8 FeNa.
non-mineral nutient regimen
(phase 1) CO2 (2%, v/v) at a flow rate of 15 l.h-1 in 300ml culture volume; (phase 4) 0% (w/v) glucose.
pH regimen
7.5
plant developmental stage
G1 phase
plant growth medium
in vitro liquid culture medium
plant structure
cell
plant treatment
photo-autotrophy
ploidy
haploid
sample material processing
9 x 108 cells were crosslinked at room temperature using 0.37% formaldehyde for 10 minutes, after which crosslinking was quenched by 0.125 M glycine for 10 minutes at room temperature. Cells were washed twice with water and centrifuged at 3000 g for 5 min at 4°C, subsequently resuspended in 200µl of Lysis Buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8, 1x complete, EDTA-free Protease Inhibitor Cocktail (Roche #04693132001)) and frozen with liquid nitrogen. Next, cells were sonicated using Bioruptor Plus with 20 cycles of 30 sec ON/OFF at 4°C to achieve DNA fragments size of approximately 200 bp and centrifuged for 5 min at 4500 g at 4°C to remove cell debris. The supernatant was diluted ten times with ChIP dilution buffer (1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8, 167 mM NaCl, complete, EDTA-free Protease Inhibitor Cocktail (Roche #04693132001)), 200µl aliquot was used for each IP incubated overnight at 4°C with 1.5µg of antibody. Antibodies specific for the following epitopes were used: H3 (Millipore/Merck, 07-690, lot #3683182), H3K4me3 (Millipore/Merck, 07-473, lot #3660317), H3K9me2 (Abcam, ab1220, lot #GR3247768-1), H3K9me3 (Active motif, 39161, lot #9919003), H3K27me1 (Sigma/Merck, 07-448, lot #3542474), H3K27me2 (Abcam, ab24684, lot GR3390336-1), H3K27me3 (Millipore/Merck, 07-449, lot #2826067; Abcam, ab6002, lot #GR3393630-1; Diagenode, C15410069, lot #A1818P). The next day, the samples were mixed with 20 µl Dynabeads Protein A (Thermo) and incubated for another 2 hours at 4°C. Next, beads were successively washed three times (5 min/wash) with Low Salt Wash Buffer (150 mM NaCl, 0.1% SDS, 1% Triton X 100, 2 mM EDTA, 20 mM Tris-HCl pH 8), High Salt Buffer (500 mM NaCl, 0.1% SDS, 1% Triton X 100, 2 mM EDTA, 20 mM Tris-HCl pH 8), LiCl Wash Buffer (0.25 M LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8) and finally with TE Buffer (10 mM Tris-HCl pH 8, 1 mM EDTA). DNA was decrosslinked, purified, and eluted using IPureKit (Diagenode).
sample name
cs_repA1
scientific_name
Chlorella sorokiniana