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Sample GSM1149448

Query DataSets for GSM1149448

Status

Public on Nov 19, 2013

Title

UH_2_reads

Sample type

SRA

Source name

Strain SMY2

Organism

Yarrowia lipolytica

Characteristics

growth condition: 1% Oxygen
genotype: UPC2 deletion

Growth protocol

Cells were grown overnight from single colonies in 5 ml YPD (1% Yeast Extract, 2% Peptone, 2% Glucose; FormediumTM) at 28°C . They were diluted in 50 ml fresh YPD to an A600 of 0.2, and grown to an A600 of 1 at 21% or 1% oxygen.

Extracted molecule

polyA RNA

Extraction protocol

mRNA purified from total RNA using oligo dT Dynabeads (Invitrogen) was fragmented to an average size of 200 nucleotides by a 5 minute heat treatment (70°C) with a buffered zinc solution (Fragmentation Reagent, Ambion). Fragmentation of mRNA was stopped using an EDTA based Stop buffer (Ambion). Fragmented mRNA was incubated with 3 μg Random Hexamer Primers at 65°C for 5 min. First strand cDNA synthesis was carried out using 1 x First Strand Buffer (Invitrogen), 10 mM DTT, 500 μM dNTP mix (Invitrogen), 20 Units RNaseOUT and 200 units SuperScriptTM II Reverse Transcriptase (Invitrogen). For strand-specific library generation, unincorporated dNTPs were subsequently removed using G-50 Micro Columns (GE Healthcare). Second strand cDNA was generated using 1x Second Strand Buffer, 300 μM dNTP mix, 2 units RNaseH, 50 units DNA Polymerase I (NEB), while a 300 μM dUTP mix was used instead of a dNTP mix for the generation of a strand specific library. This material was used for library preparation. For strand-specific libraries, DNA fragments were blunted in an End Repair reaction using T4 DNA Polymerase, Klenow DNA Polymerase and T4 Polynucleotide Kinase, after which a single ‘A’ base was added to the 3’ end using dATP and Klenow Exo Fragment. To allow multiplexing of samples 6-nt barcoded Illumina compatible adapters (AGCTAT, CGATCT, GCTAGT) were ligated to the ends of the DNA fragments, in place of the commercially supplied adapters (PMID:18794863). Fragments of approximately 200-250 base pairs were purified from a 2% Agarose Gel. The second strand containing uridine was removed by treatment with 1 unit Uracil N-glycosylase (UNG) in TE Buffer at 37°C for 15 min. In all cases, purified adapter ligated DNA templates were then amplified through PCR enrichment, using PCR primers (Illumina), a dNTP mix, Phusion Polymerase (NEB) and 13 cycles of PCR. All libraries were quantified using a Qubit® Fluorometer (Invitrogen) and assessed on a 2% agarose gel. Four libraries were generated by GATC and sequenced on an Illumina HiSeq2000

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer IIx

Description

DESeq_WN_v_UH.txt

Data processing

basecalling with CASAVA v1.8
fastq files were demultiplexed using the three barcodes (AGCTAT, CGATCT, GCTAGT) Hiseq2000 libraries were received demultiplexed
demultiplexed fastq files were aligned to the Y. lipolytica genome using TopHat 2.0.6, discarding reads that map to two or more regions and allowing one mismatch per aligned read
BAM files were converted into SAM files using SAMtools. Total counts for annotated features were calculated using HtSeq-count. These counts were used as input for DESeq. Normalization and differential gene expression comparisons were carried out using DESeq
Supplementary_files_format_and_content: 4 files normalized using DESeq for 1) wildtype normoxia vs wildtype hypoxia 2) wildtype normoxia vs upc2 deletion hypoxia 3) wildtype normoxia vs sre1 deletion hypoxia and 4) wildtype hypoxia vs sre1 deletion hypoxia

Submission date

May 28, 2013

Last update date

May 15, 2019

Contact name

Geraldine Butler

E-mail(s)

geraldine.butler@ucd.ie

Organization name

University Colege Dublin Conway Institute

Department

School of Biomolecular and Biomedical Science