SAMN37628455
External Id
The Australian National University
INSDC center name
2024-04-24T00:00:00Z
INSDC first public
2024-04-24T03:40:03.338Z
INSDC last update
SRS19033156
INSDC secondary accession
live
INSDC status
Microbe, viral or environmental
NCBI submission model
Microbe.1.0
NCBI submission package
SRS19033156
SRA accession
565 m
altitude
Nikolay Shirokikh, Protein Biosynthesis and Homeostatic Control Group, nikolay.shirokikh@anu.edu.au, The John Curtin School of Medical Research, The Australian National University, 131 Garran road, Acton, Canberra, ACT 2601, Australia
biomaterial_provider
Yoshika Janapala and Katrina Woodward
collected_by
2022-12-20
collection_date
Yeast cells of strain BY4741 exponentially growing in YPAD media at 23C were subjected to rapid media replacement by brief pelleting of the cells using centrifugation, and cell pellet resuspension in YPA media (no added glucose). Upon 10-minute incubation in YPA media at 23C, the cell cultures were snap-chilled by adding melting water ice followed by the addition of 2.2% w/v formaldehyde to stabilise translational complexes bound to mRNA. Following 10-minute incubation on ice with gentle mixing, the media was replaced with washing buffer by pelleting and re-suspending the cells. Cells were then pelleted and resuspended in a smaller volume of storage buffer, and the suspension droplet-frozen in liquid nitrogen. Cell wall disruption and lysis occurred by cryo-milling frozen cell suspension droplets, followed by the removal of large cell debris using low-speed centrifugation of the grindate to obtain clarified cell lysate. The separation of translational complexes contained in the clarified lysate then occurred using lysate ultracentrifugation through sucrose gradients. Upon ultracentrifugation, the ribosome, disome and trisome fractions of the polysomes were identified in the sedimentation profiles from the 254 nm absorption patterns. The identified light polysome (ribosome, disome and trisome) fraction was isolated and de-proteinised, while crosslinks reversed through heating. The de-blocked RNA was then purified using SPRI beads. The purified RNA was used to construct direct RNA sequencing libraries with standard Oxford Nanopore Technologies approach based on the direct RNA sequencing kit SQK-RNA002. Oligo(dT) priming was used, and the RNA strands were sequenced using MinION Mk1B device and FLO-MIN106D R9.4.1 flowcells. Data were collected using MinKNOW version 5.5.11, basecalling was performed with Guppy version 6.5.7. Basecalled reads were aligned to the Saccharomyces_cerevisiae.R64-1-1.dna_sm.toplevel.fa using minimap2 version 2.26.
description
MATa his3delta1 leu2delta0 met15delta0 ura3delta0
genotype
Australia
geo_loc_name
laboratory cell culture
host
biological replicate B
isolate
lab YPAD media
isolation_source
MATa
mating_type
Saccharomyces cerevisiae
http://purl.obolibrary.org/obo/NCBITaxon_4932
organism
1l cell culture with OD600nm of 0.7-0.8 AU
samp_size
RDT fraction of starved cells replicate B
sample_attribute
cell culture
sample_type
BY4741
strain
23 ℃
temp
Direct RNA sequencing of 10 minute glucose-starved yeast light polysome (ribosome, disome, trisome) mRNA, replicate B
title