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| Status |
Public on Dec 25, 2023 |
| Title |
In vivo sample 3 |
| Sample type |
SRA |
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| Source name |
168 wild type
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| Organism |
Bacillus subtilis |
| Characteristics |
cell type: 168 wild type
genotype: BGSC strain 1A1 wild type: trpC2
treatment: grown to OD 1.5
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| Treatment protocol |
For the in vivo reactions, cells were harvested in 25 mM Hepes-KOH pH 7.5, 100 mM KOAc, 15 mM Mg(OAc)2, 0.1% NP-40, 0.5 mM Tris Carboxy Ethyl Phosphene (TCEP) buffer supplemented with protease inhibitor cocktail (Roche), flash frozen in liquid nitrogen and lysed under cryogenic conditions using a Retsch MM400 (Retsch). The lysate was cleared at 16,000 rpm for 15 min and incubated with anti-FLAG M2 affinity beads (Merck) for 1.5 hours at 4°C on a turning wheel. After in-batch wash with 20 ml lysis buffer without protease inhibitors, the beads were transferred to a mobicol column and washed with 4 ml of 25 mM Hepes-KOH, pH 7.5, 100 mM KOAc, 15 mM Mg(OAc)2, 0.01% DDM (Dodecylmaltoside), 0.5 mM Tris Carboxy Ethyl Phosphene (TCEP) buffer after which the RNase R complexes were eluted using 0.2 mg/ml 3X FLAG peptide (Sigma) in wash buffer. To sequence the in vitro degradation reactions, 5 µM RNase R was mixed with 1,5 µM 30S subunits in a 10 µl volume at 37°C and the reaction was stopped at 4 min with the addition of 1 ml Trizol.
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| Growth protocol |
For the in vivo experiments B. subtilis 168 wild type cells (BGSC strain 1A1 wild type: trpC2) expressing RNase R-FLAG from pHT01 p43 wRBS RNase R-GS5-FLAG tGyrA were grown at 37 °C in Lysogeny broth (LB) medium (Roth) supplemented with 5 μg/mL chloramphenicol and shaking at 145 rpm until OD600 of 1.5. For the in vitro experiments BL21-CodonPlus(DE3)-RIL cells (Stratagene) transformed with expression vectors were grown in Lysogeny broth (LB) medium (Roth) supplemented with antibiotics (30 μg/mL kanamycin and 34 μg/mL chloramphenicol. His6-TEV tagged RNase R wt was expressed by induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) for 16 h at 18°C. To isolate ribosomes, the Δrnr strain (BGSC BKE33610 trpC2; Δrnr::erm) was grown in 2 L LB media until OD600= 0.8.
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| Extracted molecule |
other |
| Extraction protocol |
To extract the RNA from the Flag-IP or from the in vitro reaction 1 ml of Trizol reagent (Invitrogen) was used according to the manufacturer’s instructions with an additional 75% EtOH wash step at the end.
The samples were then taken into a modified NextFlex small RNA seq v4 protocol. Inputs were standardized to 206 ng as measured by RNA Qubit HS. Samples had 3’ adapters ligated with an adapter dilution of 1:1, followed by an adapter inactivation step (Steps A and B). Samples were cleaned up with Adapter Depletion Solution, beads, and isopro-panol following Step E but with reagent volumes adjusted for the smaller reactions. Sam-ples were resuspended in 12 µl water, and 11.2 µl were taken, added with 4 µl of 5X T4PNK buffer (NEB), and fragmented at 94°C for 1 min. 4 µl of 10 mM ATP and 0.8 µl of T4PNK were added, and samples were incubated at 37°C for 30 mins, followed by deac-tivation at 65°C for 20 mins. Then samples were taken into the 5’ ligation step from the Nextflex protocol with adapters diluted 1:3 (Step C), and the remainder of the protocol was followed as per manufacturer instructions. The positive control was amplified with 16 PCR cycles, while the DP samples were amplified with 25 PCR cycles. Samples were cleaned up individually with a 1.3x bead ratio, and checked on the bioanalyzer. Samples were pooled equimolarly, and cleaned up once more with a 1x bead ratio. Samples were sequenced using the MiSeq 50 bp v2 kit with the following read mode: 5-8-0-61. Sam-ples were demultiplexed using bcl2fastq, adapter trimming was performed with cutadapt, sequences from Read 2 were reverse-complemented (_rc) and taken forward into alignments.
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| Library strategy |
RNA-Seq |
| Library source |
other |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
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| Description |
Bacillus subtilis
read 2 sequence
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| Data processing |
The data was aligned using Novoalign (www.novocraft.com; version 3.06) to the B. subtilis 16S rRNA sequence (16S_rRNA.fs file). After generating the bam files, bed-graph files were generated using bedtools and visualized using the IGV genome brows-er.
Assembly: Bacillus/1-1554 subtilis 168 | BSU_rRNA_4 | rrnA-16S
Supplementary files format and content: BAM files of reads mapped to 16S rRNA
Supplementary files format and content: bedgraph files of reads mapped to 16S rRNA
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| Submission date |
Dec 20, 2023 |
| Last update date |
Dec 25, 2023 |
| Contact name |
Sander Granneman |
| E-mail(s) |
[email protected]
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| Organization name |
University of Edinburgh
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| Department |
Centre for Synthetic and Systems Biology
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| Lab |
Granneman lab
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| Street address |
Mayfield Road, Kings Buildings, Waddington building, room 3.06
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| City |
Edinburgh |
| ZIP/Postal code |
EH9 3JD |
| Country |
United Kingdom |
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| Platform ID |
GPL21373 |
| Series (1) |
| GSE251701 |
Structural basis of ribosomal 30S subunit degradation by RNase R |
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| Relations |
| BioSample |
SAMN38984230 |
| SRA |
SRX22985340 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7986194_invivo_3_439_1_trimmed_rc_sorted_plus_strand_3end.bedgraph.gz |
6.8 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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