ENA first public
2022-01-30
ENA last update
2022-01-30
External Id
SAMEA6601888
INSDC center alias
EI
INSDC center name
EI
INSDC first public
2022-01-30T00:13:05Z
INSDC last update
2022-01-30T00:13:05Z
INSDC status
public
Submitter Id
5e5eceac16aafb00014ce0d9:sample:Nb_gDNA_1_Plant1/2a
age
5
broker name
Collaborative Open Plant Omics broker account, Earlham Institute, Norwich
dark phase of controlled light/dark cycle
8
extraction
Before extracting DNA, plants were put in continuous darkness for three days and leaf tissue was harvested, flash frozen in liquid nitrogen, and ground to a fine powder. The tissue for this sample comes from a single plant but it was not recorded if this was individual 1 or individual 2. For high concentration, low integrity DNA (for Illumina sequencing), a modified CTAB-based protocol adapted from Michael et al. Nature communications 9(1),1-8 (2018) was used. Briefly, 5 g of frozen powdered leaf tissue was incubated with 20 mL CTAB lysis buffer (100 mM Tris-HCl, 2% CTAB, 1.4 M NaCl, 20 mM EDTA, pH 8.0) containing 20 _g/mL proteinase K for 20 minutes at 55 ¡C. Aliquots of 2.5 mL were extracted with 0.5 volumes of chloroform, mixed by inversion and centrifuged at 20,000 x g for 30 minutes at room temperature. The upper aqueous phase was mixed with an equal volume volume of 25:24:1 phenol:chloroform:isoamyl alcohol (PCI) and centrifuged at 20,000 x g for 30 minutes at room temperature. DNA was precipitated from the upper aqueous phase with 0.7 volumes (1.4 mL) ice-cold isopropanol. Precipitate was pelleted by centrifugation at 20,000 x g for 30 minutes at 4 ¡C, washed with ice-cold 70% ethanol and air-dried and room temperature, DNA was resuspended in TE buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA) containing 1.4 mg/mL RNase A at 37 ¡C with vigorous orbital shaking (300 rpm). RNase A was removed by two extractions with PCI with phases separated by centrifugation at 20,000 x g for 20 minutes at 4 ¡C. DNA was precipitated as above and resuspended in 200 µL of TE buffer. To increase the final concentration, all samples were pooled using the Zymo Research Genomic DNA Clean & Concentrator-10 column (Irvine, CA, USA) following the manufacturerÕs instructions and eluted with 50 µL DNA Elution buffer at 70 ¡C to yielding 6.5 µg of DNA (185 ng/µL)
light phase of controlled light/dark cycle
16
sample name
5e5eceac16aafb00014ce0d9:sample:Nb_gDNA_1_Plant1/2a
source name
leaf tissue
temperature
22