|
| Status |
Public on Nov 19, 2019 |
| Title |
Npun_F1710/11 overexpresser rep3 |
| Sample type |
SRA |
| |
|
| Source name |
Bacterial culture
|
| Organism |
Nostoc punctiforme PCC 73102 |
| Characteristics |
strain: ATCC 29133
alkane production: Higher than normal
|
| Treatment protocol |
The two gene Npun_F1710/11 expression (2g) plasmid was made by PCR amplifying adjacent genes Npun_R1711 and Npun_R1710 and the upstream intergenic region with subsequent cloning into pSCR119, a shuttle plasmid capable of replication in both E. coli and N. punctiforme as previously described (Peramuna, 2015).
|
| Growth protocol |
Nostoc punctiforme PCC 73102 (ATCC 29133) liquid cultures were grown in 125 mL Erlenmeyer flasks containing 50 mL of Allen and Arnon media (AA) diluted four-fold (AA/4) (Allen and Arnon, 1955) supplemented with 5 mM 3-(N-morpholino)propanesulfonic acid (MOPS), 2.5 mM NH4Cl, 2.5 mM KNO3 and 2.5 mM NaNO3 (collectively known as MAN). Plate AA media was not diluted and contained MAN and 1% Noble agar. Cultures harboring plasmid, pSCR119, were supplemented with neomycin at a final concentration of 10 μg/mL. Flasks were incubated under a white fluorescent light (12–15 μmol photons/m2/sec) at 25 °C and shaken at 120 rpm. Plates were incubated at the same illumination and temperature without shaking in a CO2 enriched (5,000 ppm) growth chamber.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Triplicate cultures of log phase wild-type plasmid-only control (WTC) and 2g strains were harvested by a 2-minute centrifugation at 6,000xg. The pellet was suspended in 700 µl of media prior to flash freezing and storage at -80°C. RNA was harvested as previously described {Schmidt-Goff, 1993 #1370}. The samples were then further purified using a RNeasy Mini Kit (Qiagen) with on-column DNase treatment. Samples were aliquoted and stored at -80°C until used. 5 µg of each sample were cleaned and concentrated further using an RNA Clean & Concentrator -5 kit (Zymo). rRNA depletion was then performed using Terminator 5’-Phosphate-Dependent Exonuclease (Epicentre) following the manufacturer’s protocol using Buffer A
RNA libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina per the manufacturer’s instructions. To determine the quality of the libraries, an Experion 1K DNA analysis kit (Bio-Rad) was used to assess the size distribution of the cDNA library following manufacturer’s instructions. Each cDNA library was quantified using a Qubit Fluorometer and Qubit dsDNA High Sensitivity reagents (Invitrogen) using manufacturer’s instructions, and also using qPCR following the protocol from the Library Quantification Kit for Illumina platforms (KAPA cat # 0796014000). Samples were normalized to 10 nM and multiplexed based on qPCR derived concentrations. The multiplexed library containing all 6 samples were sequenced on an Illumina HiSeq 2500 system where 100 bp single-end reads were generated (UC Irvine Genomics High Throughput Facility).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
High levels of alkane production
cuffdiff_gene_exp.diff.txt
|
| Data processing |
Each sample’s reads were aligned to the N. punctiforme complete genome sequence (Genbank accession number: CP001037) using Tophat software (v2.0.13; http://ccb.jhu.edu/software/tophat/fusion_index.shtml), and expression levels were quantified using Cufflinks software (v2.2.1). Finally, all the individual Cufflink outputs were merged together using Cuffmerge software (v1.0.0), and the WTC transcriptome was compared to the LD/alkane overproducer (2g) transcriptome using Cuffdiff software (v2.2.1). The output provided significant differences (q-value <0.05) between the WTC and 2g strains based on Fragments Per Kilobase of transcript per Million mapped reads (FPKM).
Genome_build: N. punctiforme complete genome sequence (Genbank accession number: CP001037)
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each sample; normalized abundance measurements output from Cuffdiff
|
| |
|
| Submission date |
Nov 18, 2019 |
| Last update date |
Nov 20, 2019 |
| Contact name |
Kerry Kevin Cooper |
| E-mail(s) |
[email protected]
|
| Phone |
520-621-3342
|
| Organization name |
University of Arizona
|
| Department |
School of Animal and Comparative Biomedical Sciences
|
| Lab |
Cooper
|
| Street address |
1117 E. Lowell St.
|
| City |
Tucson |
| State/province |
AZ |
| ZIP/Postal code |
85721 |
| Country |
USA |
| |
|
| Platform ID |
GPL27781 |
| Series (1) |
| GSE140625 |
Transcriptomic analysis of cyanobacterial alkane production |
|
| Relations |
| BioSample |
SAMN13323561 |
| SRA |
SRX7177514 |
