|
| Status |
Public on Mar 24, 2020 |
| Title |
MS2 rep3 |
| Sample type |
SRA |
| |
|
| Source name |
Bacterium_MS2
|
| Organism |
Zymomonas mobilis |
| Characteristics |
strain: 8b
plasmid: pMS2
growth phase: late exponential phase (OD600=1.0)
|
| Growth protocol |
Strains were grown in 100 mL of RMG to an OD600 of 1.0.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell pellets resuspended in 1 mL TRIzol reagent (Invitrogen) and transferred to screw cap tubes containing glass beads (Sigma) and incubated at 25°C for 5 min. Cells were lysed using a mini-beadbeater (BIOSPEC), with 100 s pulses three times. Cells were kept on ice for 10 min between each 100 s treatment. The beads and cellular debris were centrifuged at 4°C for 2 min. The supernatant was transferred to a clean siliconized 2 mL tube. After addition of 300 µL of chloroform:isoamyl alcohol mix (v/v 24:1), the samples were inverted for 15 s, and then incubated at 25°C for 3 min. Then, tubes were centrifuged at 13,000 rpm for 10 min and the aqueous top phase transferred to a clean siliconized 1.5 mL tube. Following this step, 270 µL of isopropanol and 270 µL of a mixture of 0.8 M sodium citrate and 1.2 M sodium chloride was added. The samples were mixed well, and then incubated on ice for 10 min. The RNA was pelleted by centrifugation at 13,000 rpm for 15 min. The pellet was washed with 1 mL 95% cold ethanol and centrifuged for 5 min. The pelleted RNA was allowed to air-dry for 5 min, and was resuspended in 50 µL RNase-free water (Ambion). RNA was digested with DNase I (RNase-free, ThermoScientific) for 1 h at 37°C to prevent genomic DNA contamination. DNase I was heat-inactivated by adding 0.5 mM EDTA to the reaction mixture and incubating at 75°C for 10 min. Then, RNA was incubated with isopropanol and GlycoBlue™ (ThermoScientific) at -20°C overnight. After centrifugation, pelleted RNA was washed with 95% cold ethanol and centrifuged. RNA was resuspended in 50 µL RNase-free water (Ambion) and stored at -80°C before sequencing.
NEBNext® Multiplex RNA Library Prep Set for Illumina® (New England Biolabs Inc.) was used for generating RNA libraries.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
2MS2-cont3_S45
|
| Data processing |
Illumina NextSeq 500 Real-Time Analysis (RTA v2) basecalling
Adapter sequences and low-quality ends (phred quality < 30) were trimmed from the raw fastq files with cutadapt (v1.3) and reads shorter than 22 nt were discarded.
FastQC was used to verify good read quality for the trimmed files.
Reads were aligned to the Z. mobilis ZM4 reference genome (taxonomy ID 264203) with BWA-mem (v0.7.12-r1039).
Aligned reads were filtered for quality (MAPQ ≥ 10) and sorted by chromosomal coordinates using SAMtools (v0.1.19-44428cd).
The number of reads aligned to each gene was calculated using HTSeq (intersection-nonempty mode for overlaps).
DESeq2 was used to normalize and identify differentially expressed transcripts between strains.
Genome_build: NC_006526.2
Supplementary_files_format_and_content: tab-delimited text file of normalized reads for each gene by sample
|
| |
|
| Submission date |
Nov 20, 2017 |
| Last update date |
Mar 24, 2020 |
| Contact name |
Lydia M Contreras |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Texas at Austin
|
| Department |
McKetta Department of Chemical Engineering
|
| Street address |
200 E. Dean Keeton St Stop C0400
|
| City |
Austin |
| State/province |
TX |
| ZIP/Postal code |
78712 |
| Country |
USA |
| |
|
| Platform ID |
GPL24294 |
| Series (2) |
| GSE107192 |
MS2-affinity purification coupled to RNA-sequencing (MAPS) reveals targetomes of Z. mobilis sRNAs Zms4 and Zms6 |
| GSE107219 |
Mapping regulatory networks of Zymomonas mobilis small RNAs Zms4 and Zms6 |
|
| Relations |
| BioSample |
SAMN08046663 |
| SRA |
SRX3411513 |
