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| Status |
Public on Jul 08, 2020 |
| Title |
4B4 rep1 |
| Sample type |
SRA |
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| Source name |
bacterial cell
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| Organism |
Lactococcus cremoris |
| Characteristics |
strain: 4% isobutanol tolerant
culture: late log phase
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| Growth protocol |
4B4 was grown in GM17 media (containing 4% isobutanol) in 30 degrees incubator under static conditions to late log phase, cells were arrested and proceeded for RNA isolation
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by slight modification of the method described by(Dijkstra et al., 2016) for L. lactis. Cells from the late-log phase were snap frozen and harvested at (5000 x g, 10 mins, 4 degrees). Cell pellets were resuspended in 500ul of ice-cold TE buffer pH 7.5 and transferred to ice-cold 2ml microfuge tube (Eppendorf), after which 500 μl phenol/chloroform mixture (1:1), 30 μl 10% SDS, 30 μl 3 M sodium acetate (pH 5.2), and 500 mg glass-beads (150-212 μm, Sigma-Aldrich, St. Louis, USA) were added. The mixture was vortexed for 4 minutes at maximum speed and then 400 μl of chloroform isoamyl alcohol (24 :1) was added. The mixture was spun at 10000 x g at 20 degrees for 2 minutes. The supernatant was transferred to fresh microfuge tube and equal volume of RNAzol (Sigma) was added and steps were followed according to manufacturer’s protocol. The total RNA concentration and quality was determined by Agilent bioanalyzer 2100 expert.
Illumina HiSeq 2000/2500 Paired end run; 2 X 100 bp 30 million reads (3GB) per sample. ~3 ug of total RNA was used to prepare the RNA seq library. In short, mRNA molecules were purified using magnetic beads. Folllowing purification, mRNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were used to synthesize first strand cDNA using reverse transcriptase and random primers followed by second strand cDNA synthesis using DNA polymeraseI and RNase H. These cDNA fragments were used to generate RNA libraries. Bioanalyzer plots were used at every step to assess mRNA quality, enrichment success, fragmentation sizes,and final library sizes. The size distribution of the sequencing library was determined by AgilentTapestation or Bioanalyzer. After the libraries were constructed, paired end run were performed on Illumina’s HiSeq 2500 platform to obtain 2 x100bp reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Illumina HiSeq 2500 reads were trimmed for removing adapter sequences using Trimmomatic(v.0.36). Reads quality was checked for each sample using FastQC v0.10.1. Reference genome (Fasta) and annotation file (Gff) of L. lactis NZ9000 was procurred from Ensembl and Gff annotation file was converted to Gtf using "Gffread" from Cufflinks Suite and then reference indices were created using Bowtie2. BAM files generated by Bowtie2 tool alfter alignment were then used to calculate mapped reads abundance using RSEM. Normalization was done to rule out the effect of library size and reads length by estimating FPKM values for paired-end reads for each sample. Differential expression was performed using edgeR, which is based on negative binomial distribution. We selected the genes having at least two log fold change values and false-discovery-rate (FDR) corrected P value < 0.05 to be considered as differentially expressed genes.
Genome_build: Lactococcus_lactis_subsp_cremoris_nz9000.ASM14320v1
Supplementary_files_format_and_content: Exp_sample.genes.results represents output generated from RSEM. The abundance of each transcript is represented by its corresponding FPKM values.
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| Submission date |
Mar 27, 2018 |
| Last update date |
Jul 08, 2020 |
| Contact name |
Krishna Jyoti Mukherjee |
| E-mail(s) |
kjmukherjee@mail.jnu.ac.in
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| Phone |
01126704087
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| Organization name |
JNU
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| Department |
SBT
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| Lab |
Bioprocess and Biosystems engineering lab
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| Street address |
Behind administration building
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| City |
NewDelhi |
| State/province |
Delhi |
| ZIP/Postal code |
110067 |
| Country |
India |
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| Platform ID |
GPL24381 |
| Series (1) |
| GSE107996 |
Designing of Lactococcus lactis platform for isobutanol production using multiple rounds of adaptive laboratory evolution |
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| Relations |
| BioSample |
SAMN08800371 |
| SRA |
SRX3852246 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3068949_Expression_4B4.genes.results.txt.gz |
42.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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