Gene Expression Nebulas
A data portal of transcriptomic profiles analyzed by a unified pipeline across multiple species

Gene Expression Nebulas

A data portal of transcriptome profiles across multiple species

PRJNA605000: Adapting the 10x Genomics Platform for single-cell RNA-seq in yeast reveals the importance of stochastic gene expression during the lag phase

Source: NCBI / GSE144820
Submission Date: Feb 05 2020
Release Date: Feb 06 2020
Update Date: Jun 08 2020

Summary: Isogenic population of yeast cells was grown in a maltose-glucose-maltose media shift schema. Time point samples were taken throughout the experiment, and were analyzed for single-cell gene expression using 10x Genomics platform.

Overall Design: Yeast cells experience a halt in growth after glucose-maltose shift, aka lag phase. Two samples are taken during glucose growth. Two samples are taken during lag phase in maltose. One sample comprises a mix of cells actively grown on glucose and cells actively growing in maltose. This last sample serves as a control to see if we can detect the expected population structure.

GEN Datasets:
GEND000326
Strategy:
Species:
Healthy Condition:
Cell Line:
Development Stage:
Protocol
Growth Protocol: Cells were grown in rich media with either glucose (YP-glucose-5%) or maltose (YP-maltose-10%) in turbido-stat conditions. The cell counts were kept below 10 million cells per ml.
Treatment Protocol: The cells were initially inoculated in YP-maltose-10% and grown in turbido-stat for 3 overnights. Afterwards they were washed into glucose media (3x, 5min, 3000rpm). After 6h of growth in glucose, the first sample was taken (glu_6h). After 12h of glucose growth the 2nd sample was taken (glu_12h). Afterwards the cells were washed into maltose media, were they cease to grow and enter lag phase. 1h after the shift to maltose the 3rd sample was taken (in_lag_1h). After 3h in lag phase the 4th sample was taken (in_lag_3h). The 5th sample is a 50-50 mix of cells from two growth stages: A part of it is the cells from maltose pregrowth, and another part is the cells from glu_12h sample.
Extract Protocol: We used the Chromium Single Cell 3′ kit (v2) of 10x Genomics for scRNA-seq. We modified the protocol to include zymolyase for digestion of the cell wall. The cells were pre-grown in 3 mL maltose media (YPMAL10%) for two overnights in dilute conditions. The media was refreshed after the first overnight and at each step serial dilutions were made to capture cultures with a cell count of ~2*106 cells/mL. At the end of maltose pre-growth, the cells were washed and inoculated into 50 mL glucose media to reach a starting cell density of 104 cells/mL. The cells were then grown in glucose for 12 hr, before being washed to maltose media where they experience a lag phase.
Library Construction Protocol: 10x Genomics, V2 chemistry, For each sequencing run (1 NextSeq & 1 HiSeq with 2 lanes) and each sequencing lane and each sample, three fastq files are provided. The first fastq file corresponds to i7 index (I1, 8bps) and indicates the sample index. The second fastq files corresponds to read 1 (R1, 26bps) and indicates the cell barcode and Unique Molecular Identifier (UMI). The 3rd fastq file corresponds to Read 2 (R2, 98 bps) and corresponds to cDNA sequence.
Sequencing
Molecule Type: poly(A)+ RNA
Library Source:
Library Layout: PAIRED
Library Strand: -
Platform: ILLUMINA
Instrument Model: Illumina HiSeq 3000
Strand-Specific: -
Samples
Basic Information:
Sample Characteristic:
Biological Condition:
Experimental Variables:
Protocol:
Sequencing:
Assessing Quality:
Analysis:
Data Resource GEN Sample ID GEN Dataset ID Project ID BioProject ID Sample ID Sample Name BioSample ID Sample Accession Experiment Accession Release Date Submission Date Update Date Species Race Ethnicity Age Age Unit Gender Source Name Tissue Cell Type Cell Subtype Cell Line Disease Disease State Development Stage Mutation Phenotype Case Detail Control Detail Growth Protocol Treatment Protocol Extract Protocol Library Construction Protocol Molecule Type Library Layout Strand-Specific Library Strand Spike-In Strategy Platform Instrument Model Cell Number Reads Number Gbases AvgSpotLen1 AvgSpotLen2 Uniq Mapping Rate Multiple Mapping Rate Coverage Rate
Publications
A new protocol for single-cell RNA-seq reveals stochastic gene expression during lag phase in budding yeast.
eLife . 2020-05-18 [PMID: 32420869]