|
| Status |
Public on Feb 19, 2021 |
| Title |
L1900796_Nr_1_CoIP_RSP_6037 |
| Sample type |
SRA |
| |
|
| Source name |
wildtype
|
| Organism |
Cereibacter sphaeroides 2.4.1 |
| Characteristics |
strain: 2.4.1
plasmid: pRK6037_3xFLAG
|
| Treatment protocol |
Cells were harvested during exponential growth phase. Cell-pellets were resuspended in 2 ml of cold lysis buffer (20 mM Tris pH 7.5, 150 mM KCl, 1 mM MgCl2, 1 mM DTT) and disrupted by sonication (Pfeiffer, 2007). Cell lysate was centrifuged for 10 min at 13000 rpm and 4°C, followed by a ultra-centrifugation step (100000 rpm, 1 h, 4°C). Afterwards the supernatant was mixed with 40 µl of ANTI-FLAG M2 Magnetic Beads (Sigma-Aldrich) and incubated for 2 h at 4°C under rotation. Following five washing steps with 500 µl of lysis buffer, magnetic beads were resuspended in 500 µl of lysis buffer and RNA was isolated with phenol and chloroform-isoamylalcohol before precipitation with 1/10x vol. 3 M sodium acetate pH 4.5 and 2.5x vol. 96% Ethanol overnight.
|
| Growth protocol |
All cultures were grown in malate minimal medium under microaerobic conditions (25 μM dissolved oxygen) at 32°C and 140 rpm in the dark.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the phenol method and remaining DNA was removed by a DNaseI treatment (Invitrogen).
RNA libraries were prepared for sequencing using standard Illumina protocols
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
All reads were trimmed for quality and adaptor sequences using trimmomatic
Reads were mapped to the Rhodobacter sphaeroides 2.4.1 whole genome (NC_007493.2, NC_007494.2, NC_009007.1, NC_007488.2, NC_007489.1, NC_007490.2, and NC_009008.1) using the READemption pipeline (version 0.4.3) subcommand align with the integrated mapper segemehl (version 0.2.0)
READemption (version 0.4.3) was used for gene quantification, subcommand gene_quanti
READemption (version 0.4.3) with DESeq2 (version 1.22.1) was used to perform the differential gene expression analysis, subcommand deseq
READemption (version 0.4.3) was used to compute the coverage files, subcommand coverage
Genome_build: GCF_000012905.2 ASM1290v2
Supplementary_files_format_and_content: csv contains gene quantification and DESeq2 analysis
|
| |
|
| Submission date |
Feb 10, 2020 |
| Last update date |
Feb 19, 2021 |
| Contact name |
Gabriele Klug |
| E-mail(s) |
Gabriele.Klug@mikro.bio.uni-giessen.de
|
| Organization name |
Justus-Liebig University Gießen
|
| Department |
Molecular- and Microbiology
|
| Street address |
Heinrich-Buff-Ring 26-32
|
| City |
Gießen |
| ZIP/Postal code |
35392 |
| Country |
Germany |
| |
|
| Platform ID |
GPL28141 |
| Series (1) |
| GSE145045 |
Co-immunoprecipitation of the small DUF1127 protein RSP_6037xFLAG and pRK0557_3xFLAG for RNA interaction |
|
| Relations |
| BioSample |
SAMN14079053 |
| SRA |
SRX7703392 |
