ENA first public
2022-01-30
ENA last update
2022-01-30
External Id
SAMEA6601890
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
5e5ece6e16aafb00014ce0d6:sample:Nb_gDNA_12_Plant9
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 from a single unique plant, flash frozen in liquid nitrogen, and ground to a fine powder. For high concentration, high integrity DNA (for Oxford Nanopore), a modified CTAB-based protocol adapted from Michael et al. Nature communications 9(1),1-8 (2018) was used. Briefly, 1.0 g of frozen powdered leaf tissue was mixed with 3 mL CTAB lysis buffer containing 20 μg/mL proteinase K and incubated at 55 °C at 500 rpm. After 15 minutes, 20 µL of 100 mg/mL RNaseA was added and incubation continued for 15 minutes before a further 20 µL of 100 mg/mL RNaseA was added. After 15 minutes, the mixture was chilled on ice for five minutes. Extractions with 0.5 volumes of chloroform and equal volumes of PCI were performed as above except that wider-bore pipette tips were used at all stages. DNA was precipitated and resuspended as above in 25 µL TE buffer. Yield (8-21 µg at 350-900 ng/µL) was determined using a Qubit fluorometer (ThermoFisher, Waltham, MA, USA). Fragments of >40 kb were collected for library preparation using BluePippin pulsed-field electrophoresis (Sage Science, Beverly, MA, USA). DNA was not frozen but stored at 4 °C overnight before proceeding directly to library preparation.
light phase of controlled light/dark cycle
16
sample name
5e5ece6e16aafb00014ce0d6:sample:Nb_gDNA_12_Plant9
source name
leaf tissue
temperature
22