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Sample GSM1717186

Query DataSets for GSM1717186

Status

Public on Aug 20, 2016

Title

RNA-seq EE rep2

Sample type

SRA

Source name

Kluyveromyces marxianus_early exponential

Organism

Kluyveromyces marxianus

Characteristics

strain: marxianus ATCC 36907 (KM7)
growth phase: early exponential

Biomaterial provider

Korean Collection for Type Culture (KCTC)

Growth protocol

K. marxianus var. marxianus ATCC 36907 (KM7) were obtained from Korean Collection for Type Culture (KCTC) and were grown in YBN-u media (0.67 % yeast nitrogen base without amino acids, uracil deprived amino acids and 2 % w/v dextrose) at 30 °C in a shaking incubator. They were sampled at three time points of early-exponential (EE; OD =~ 3), mid-exponential (ME; OD =~ 7), and late-exponential (LE; OD =~ 10) conditions with two biological replicates.

Extracted molecule

total RNA

Extraction protocol

Cells were harvested by centrifugation and resuspended with lysis buffer, 20 mM Tris‐HCl (pH 7.5), 140 mM NaCl, 5 mM MgCl2, and 1% Triton‐X of 300 ul. Further, the resuspended cells were lysed with 1 mL TRIzol (Invitrogen) and incubated for 5 min at room temperature. After centrifuge for 15 min at 3000 rpm, the supernatant was transferred new tube and mixed with chloroform of 200 μl for 2‐3 min. After centrifugate for 15 min, the supernatant was mixed with 3 fold of 100% ethanol or equal fold of isopropanol, 2 μl glycogen, 3M sodium acetate. After centrifuge and resuspension, prepared RNA was washed with 70 % ethanol. After drying, RNA was resuspended to DEPC-treated water. To confirm the quality of extracted RNAs, total RNA was visualized using agarose gel electrophoresis.
To remove genomic DNA, the isolated RNA was incubated at 37 °C for 1 h with 4 U of rDNase I (Ambion) and 5 μl of 10× DNase I buffer (Ambion). The DNA-free RNA was purified by phenol-chloroform extraction and ethanol precipitation. Ribosomal RNA (rRNA) was removed by using Ribo-Zero Magnetic Gold Kit (Human/Mouse/Rat) (Epicentre) according to the manufacturer’s instructions. Ribosomal RNA-depleted RNAs were checked for quality control with Agilent 2200 TapeStation system (Agilent Technologies). 200 ng mRNA was then fragmented by incubation at 70 °C for 5 min with 10× Fragmentation buffer (Ambion). The reaction was terminated by adding 1 μl of Stop solution (Ambion) and the fragmented mRNA was purified by ethanol precipitation. For first strand cDNA synthesis, 3 μg of Random primers (Invitrogen) were added to the fragmented mRNA and denatured by incubation at 65 °C for 5 min. Then the following was added to the reaction: 2 μl of 10× RT buffer (Invitrogen), 1 μl of 10 mM dNTP mix, 4 μl of 25 mM MgCl2, 2 μl of 100 mM DTT, 1 μl of SuperScript III Reverse Transcriptase (200 U/μl, Invitrogen), and 1 μl of RNaseOUTTM (40 U/μl, Invitrogen). The mixture was incubated 10 min at 25 °C for annealing then 50 min at 50 °C for reverse transcription. The reaction was terminated by incubation at 85 °C for 5 min. Synthesized first strand cDNA was mixed with 3 fold of 100% ethanol, 2 μl glycogen, 3M sodium acetate and centrifuged and resuspended with RNase and DNase free water after drying. The following mixture was added to the purified cDNA for second strand synthesis: 1 μl of 10× RT buffer (Invitrogen), 0.5 μl of 25 mM MgCl2, 1 μl of 100 mM DTT, 2 μl of 10 mM mixture of each dNTP (dATP, dGTP, dCTP, and dUTP), 15 μl of 5× second-strand buffer (Invitrogen), 5 μl of Escherichia coli DNA polymerase (10 U/μl, Invitrogen), 1 μl of E. coli DNA ligase (10 U/μl, Invitrogen), and 1 μl of E. coli RNase H (2 U/μl, Invitrogen). The mixture was incubated at 16 °C for 2 h and synthesized cDNA was mixed with 3 fold of 100% ethanol, 2 μl glycogen, 3M sodium acetate and centrifuged and resuspended with RNase and DNase free water after drying. The libraries for Illumina sequencing were constructed using TruSeqTM DNA Sample Prep Kit (Illumina Inc.) according to the manufacturer’s instructions. Briefly, the synthesized cDNA was end-repaired and 3’-ends of the blunt fragments were adenylated for the adapter ligation. The adenylated DNA fragments were ligated with Illumina adapters. A fraction of the adapter-ligated DNA between 180 bp and 380 bp was size-selected from a 2% agarose gel after electrophoresis. Size-selected DNA was purified by using MinElute Gel Extraction Kit (Qiagen) according to manufacturer’s instructions and eluted in 1× TE buffer with low EDTA (10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA) for the following enzyme reaction. For degradation of the second strand which contains dUTP instead of dTTP, 1 U of USER enzyme (NEB) was added to the purified DNA and incubated at 37 °C for 15 min. After 5 min incubation at 95 °C for enzyme inactivation, the library was enriched by PCR. The amplification was monitored on a CFX96TM Real-Time PCR Detection System (Bio-Rad) and stopped at the beginning of the saturation point. The amplified library was purified by using Agencourt AMPure XP beads and quantified using a Qubit 2.0 fluorometer (Invitrogen).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina MiSeq

Data processing

Basecalls performed using CASAVA version 1.4
Sequencing reads were mapped to the genome sequence from NCBI (AKFM00000000.1) using CLC genomics workbench (default parameter) after trimming of low quality regions [12]. RNA‐seq depth profiles are visualized using SignalMap (NimbleGen).
To obtain transcriptional units, we identified transfrags through pooling RNA‐seq libraries replicates for all three conditions. We merged nearby transfrags to reduce over-fragmentation if their distance is shorter than 40 bp but didn’t merge if their distance is longer than 100 bp to maximize median length ratio between sense transfrags and antisense transfrags. For the nearby transfrags of distance range from 40 bp to 100 bp, they are merged if p‐value of Wilcoxon rank test with two‐sided are less than 10‐20 which suggests that RNA‐seq profile of the two transfrags is statistically not different. Finally, we chose transfrags with high transcriptional expression level from DESeq analysis is over 25 percentile transcriptional level. Predicted transfrags were classified into sense (sense to ORF), antisense (antisense to ORF), and intergenic (intergenic of ORFs). If a transfrag covers more than two gene annotations, it is divided into multiple transfrags corresponding to sense, antisense, and intergenic transfrags. This process was conducted by in‐house script and manual inspection was followed. CPAT was used for the calculation of coding potential of transfrags and used for the selection of non-coding RNAs. Thus, we classified transfrags into five RNA classes by using transfrag size, coding potential, and location relative to gene annotation; mRNA (sense to protein coding genes and CPAT >= 0.364), lancRNA (antisense to protein coding genes, size >= 200 nt, and CPAT < 0.364), lincRNA (intergenic, size >= 200 nt, and CPAT < 0.364), sancRNA (antisense to protein coding genes, size < 200 nt, and CPAT < 0.364), and sincRNA (intergenic, size < 200 nt, and CPAT < 0.364). Differentially expressed transfrags were conducted by using DESeq. In KEGG enriched pathway analysis, pathways that have p‐value lower than 0.01 by two‐tailed Fisher exact test are considered to be enriched with statistical significance.
The expression levels were based upon DESeq method.
Genome_build: AKFM00000000.1
Supplementary_files_format_and_content: gff and text file with expression levels from DESeq.

Submission date

Jun 22, 2015

Last update date

May 15, 2019

Contact name

Byung-Kwan Cho

Organization name

KAIST

Lab

Systems and Synthetic Biology Lab

Street address

291 Daehak-ro