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| Status |
Public on Nov 24, 2020 |
| Title |
Trx-iCLIP (neg.ctr.) rep2 |
| Sample type |
SRA |
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| Source name |
Trx-iCLIP (neg.ctr.)
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| Organism |
Saccharolobus solfataricus |
| Characteristics |
ip: Trx
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| Treatment protocol |
Individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP) analyses were performed as described in König et al. (2010) (PMID: 20601959), with the following alteration that were necessary for the application in S. solfataricus. Cell pellets were washed with PBS, and 0.8 g were resuspended in 40 ml PBS at 4°C, and spread on a large petri dish swimming on an ice-water bath. The cell suspension was crosslinked four times at 254 nm, 300 mJ/cm², cells were mixed in between. Crosslinked cells were harvested as before and stored at -80°C.
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| Growth protocol |
S. solfataricus P2 was grown as previously described in a 10 l bioreactor (Evguenieva-Hackenberg et al., 2002) (PMID: 12359717). Cells were cultivated until an OD600 of 0.8 and harvested by centrifugation at 6,000 g and 4 °C for 10 min.
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| Extracted molecule |
total RNA |
| Extraction protocol |
For cell lysis, 0.5 g of cells were thawed on ice, resuspended in lysis buffer (10 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl2, 10% Glycerol, 0.1% Nonidet P40, 1 mM PMSF) and lysed by sonication (five 30 sec cycles, 70%). Cells debris was removed by centrifugation, and supernatant was DNase- and RNase-treated. 10x RQ1 DNase buffer was added to 1x final concentration, and 1:500 vol. Turbo-DNase (Ambion), 1:1,000 vol. RNaseOUT (Thermo Fisher Scientific) and different dilutions of RNase I (Ambion), in RQ1-buffer (high RNase: 1:1,000 vol. and low RNase 1:10,000 vol.). Extracts were incubated for 6 min at 37°C in a shaking water bath. All following steps were performed on ice / at 4°C. Immunoprecipitation was performed as described in Walter et al. (2006) (PMID: 17078816): 4.5 ml of lysate was incubated with Protein-A-Sepharose beads coupled to 100 μl of polyclonal antibody raised against His-tagged Rrp41- or Rrp4-: against His-tagged Rrp41 and Rrp4 (Witharana et al., 2012) (PMID: 22503705), or polyclonal antibody raised against Thioredoxin (Trx) from the alphaproteobacterium R. sphaeroides (Li et al., 2003) (PMID: 12624204) as a negative control for 2 h. Beads were washed ten times with high salt wash buffer (10 mM Tris pH 7.5, 1 M NaCl, 5 mM MgCl2, 10% Glycerol, 0.1% Nonidet P40, 1 mM PMSF). To remove excess of salt, beads were then washed two times with PNK-buffer (70 mM Tris pH 7.5, 10 mM MgCl2, 0.05% NP-40). All following steps were performed as described in König et al. (2010). In brief: immunoprecipitated crosslinked RNA-protein complexes were subjected to several enzymatic reactions on-bead. Subsequent de-phosphorylation of RNA 3’-ends by phosphatase-treatment, a 3’-RNA linker ligation and ³²P-5’-end labelling of the RNA using T4 polynucleotide kinase and gamma-[³²P]-ATP were performed. Complexes were resolved on a denaturing neutral-pH SDS-polyacrylamide gel electrophoresis (NuPAGE, Invitrogen), and transferred to a nitrocellulose membrane Protein-RNA-complexes were visualized by autoradiography on an x-ray film at -80°C. Complexes of adequate size were excised from the membrane and RNA was isolated by proteinase K treatment.
iCLIP library preparation was performed as described in König et al. (2010), and sequencing on an Illumina MiSeq system, 75 bp single-read. Following oligonucleotides were used: 3’-RNA linker (L31): P-UGAGAUCGGAAGAGCGGUUCAG-Puromycin Reverse transcription primers (containing random and experimental barcode,): R1clip P-NNAACCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC R6clip P-NNCCGGNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC R9clip P-NNGCCANNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC R10clip P-NNGACCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC R13clip P-NNTCCGNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC R14clip P-NNTGCCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Description |
crosslinked and co-precipitated RNA
none
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| Data processing |
demultiplex, quality filter: fastx toolkit (v0.0.14)
alignment: bowtie2 (v2.3.4) –sensitive
coverage: samtools, bedtools
Genome_build: ensembl Sulfolobus_solfataricus_p2.ASM700v1:
Supplementary_files_format_and_content: coverage: wig
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| Submission date |
Apr 22, 2020 |
| Last update date |
Nov 24, 2020 |
| Contact name |
Gabriele Klug |
| E-mail(s) |
Gabriele.Klug@mikro.bio.uni-giessen.de
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| Organization name |
Justus-Liebig University Gießen
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| Department |
Molecular- and Microbiology
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| Street address |
Heinrich-Buff-Ring 26-32
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| City |
Gießen |
| ZIP/Postal code |
35392 |
| Country |
Germany |
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| Platform ID |
GPL28445 |
| Series (2) |
| GSE149140 |
iCLIP analysis of RNA substrates of the archaeal exosome (iCLIP) |
| GSE149143 |
iCLIP analysis of RNA substrates of the archaeal exosome |
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| Relations |
| BioSample |
SAMN14670172 |
| SRA |
SRX8156862 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4490920_BC6_for.wig.gz |
55.2 Kb |
(ftp)(http) |
WIG |
| GSM4490920_BC6_rev.wig.gz |
47.8 Kb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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