Severe acute respiratory syndrome-related coronavirus
Protocols: For swab samples (\"B117_01-12\" and \"B177_01-12\"), nasopharyngeal swabs were collected into viral transport medium, which was subsequently lysed by adding 3 volumes Trizol LS. For cell samples (\"Vero_Eng02, Vero_B117, Vero_B1351\") VeroE6 cells were infected with indicated variants at a high (>1) MOI and harvested after 24 hours by lysis of the cell monolayer with trizol.  RNA was extracted from Trizol using the Zymo Direct-zol RNA miniprep kit.  PCR amplicons were generated using promega go-taq using primers against the 5' leader and within the 3' end of the N gene. End preparation was carried out using the NEBNext Ultra II End Repair A-Tailing Module (NEB). The reaction was carried out at 20 ºC for 25 minutes, followed by 15 minutes at 65 ºC to inactivate the enzyme. Samples were directly ligated with an individual barcode from the ONT Native Barcoding kit (EXP-NBD196) using NEBNext Blunt/TA Ligase Master Mix (NEB). The ligation reaction was carried out for 20 minutes at 20 ºC followed by 10 minutes at 70 ºC. After barcoding, samples were pooled with equal volumes. The pool is cleaned up using a 0.4x ratio of SPRIselect magnetic beads (Beckman Coulter). Fragment-bound beads were washed twice with Short fragment buffer (SFB) (ONT), and once with 80% ethanol. The DNA target was eluted with Buffer EB (Qiagen). To allow Nanopore sequencing, the AMII adapter (ONT) was ligated on using NEBNext Quick Ligation Module (NEB), with a 20 minute incubation at 20 ºC. A second SPRIselect bead cleanup was carried out with a 1x ratio of beads and two washes with SFB only. The final pool was eluted in Elution Buffer (ONT) and quantified by Qubit HS dsDNA assay (Thermo Fisher).
ENA first public
2024-01-01
INSDC center name
The Francis Crick Institute
INSDC status
public
Submitter Id
E-MTAB-14681:B177_10
broker name
ArrayExpress
collection date
not collected
genotype
G28881, G28882, G28883
geographic location (country and/or sea)
not collected
isolate
not applicable
sample name
E-MTAB-14681:B177_10
scientific_name
Severe acute respiratory syndrome-related coronavirus