 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 06, 2024 |
| Title |
C. botulinum IStron total RNA-seq, heterologous |
| Sample type |
SRA |
| |
|
| Source name |
BL-21
|
| Organism |
Escherichia coli |
| Characteristics |
cell line: BL-21
|
| Growth protocol |
E. coli str. BL21 (sSL0026) was transformed with plasmids encoding CboIStron with mutant tnpAS (pSL5878), CseIStron-1 (pSL6566) or CseIStron-2 with frameshifted tnpAS (pSL6630). Single colonies were inoculated in liquid LB with spectinomycin (100 µg mL−1) and grown overnight. The next day, the culture was used to inoculated 50 mL of liquid LB with spectinomycin (100 µg mL−1) and IPTG (0.1 mM) at 100× dilution and grown until OD600 reached 0.5. 1 ml of the culture was centrifuged at 6,000 rpm for 5 min and the resulting pellet was used for RNA extraction. A Clostridia strain encoding IStrons with ~80% similarity to the CboIStron experimentally characterized in this study was obtained from ATCC (strain 25772), where it was defined as belonging to an unknown species classification. Internal rRNA phylogenetic analysis led to the assignment of this strain as a member of species senegalense. C. senegalense was cultured from a lyophilized ATCC pellet in 5 mL of Gifu Anaerobic Medium Broth, Modified (mGAM; HyServe) in an anaerobic chamber (5% H2, 10% CO2 and 85% N2). All media was pre-reduced for ~24 hours before use in culturing. C. senegalense was then banked as a glycerol stock (final concentration 20%) and sub-cultured into 100 mL cultures of mGAM. The growth of these cultures was monitored with a spectrophotometer over ~6 h until a final OD600 of 0.4-0.6 was reached (exponential phase), at which point cultures were poured into two 50 mL conical tubes and cooled on ice for 10 min. The cultures were then centrifuged at 4,000 g for 10 min at 4 °C, the supernatant was decanted, and cell pellets were flash frozen in liquid nitrogen. Pellets were stored at -80 °C until RNA extraction and processing.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA from E. coli was extracted by resuspending cell pellet in 1 ml Trizol and incubating at room temperature for 10 min before adding 200 ul chloroform. The tubes were mixed and centrifuged at 12,000 g for 15 min at 4 °C. The aqueous phase was transferred to a new tube and purified using Monarch RNA Cleanup Kit (NEB). RNA was stored at -80 °C until library preparation. RNA from C. senegalense cell pellets were extracted in 96-well format using a silica bead beating-based protocol adapted from a prior study Ji et al., 2019, Nat Methods. Briefly, 200 µL of 0.1 mm zirconia silica beads (Biospec,) were added to each well of 96-well deep-well plates (Thermo Fisher Scientific). Next, cell pellets were resuspended in 500 µL DNA/RNA shield buffer (Zymo Research) and transferred to separate wells, and the plates were affixed with a sealing mat and centrifuged for 1 min at 4,500 g. To avoid overheating, the plates were vortexed for 5 s and incubated at −20 °C for 10 min before beating. Then, plates were fixed on a bead beater (Biospec) and subjected to bead beating for 5 min, followed by a 10 min cooling period. The bead beating cycle was repeated three times, and plates were then centrifuged at 4,500 g for 5 min to remove cell debris. Next, 60% of the bead beating volume was transferred to the Quick-RNA Miniprep Plus kit (Zymo Research), and RNA was purified using the manufacturer’s protocol for gram positive bacteria. RNA quality was assessed using the 260/280 nm ratio (~2.0) as measured by Nanodrop, and concentration was measured by the Qubit RNA High Sensitivity Assay Kit (Thermo Fisher Scientific) using the manufacturer’s protocol. RNA was stored at -80 °C until library preparation.
For total RNA-seq library preparation of samples from E. coli, 200 ng of purified total RNA was depleted for ribosomal RNA with Ribo-Zero Plus rRNA Depletion kit (illumina) following the manufacturers protocol and used for downstream library preparation. For samples from C. senegalense, 10 µg of purified RNA was treated with Turbo DNase I (Thermo Fisher Scientific) for 1 h at 37 °C using the manufacturer’s protocol. A 2× volume of Mag-Bind TotalPure NGS magnetic beads (Omega) was added to each sample, and the RNA was purified using the manufacturer's protocol. The RNA was then diluted in NEBuffer2 (NEB) and fragmented by incubating at 92 °C for 1.5 min. To generate RNA with 5′-monophosphate and 3′-hydroxyl ends, samples were treated with RppH (NEB) supplemented with SUPERase•In RNase Inhibitor (Thermo Fisher Scientific) for 30 min at 37 °C, followed by T4 PNK (NEB) in 1× T4 DNA ligase buffer (NEB) for 30 min at 37 °C. Samples were column-purified using RNA Clean & Concentrator-5 (Zymo Research), and the concentration was determined using the DeNovix RNA Assay (DeNovix). Illumina adapter ligation and cDNA synthesis were performed using the NEBNext Small RNA Library Prep kit. Dual index barcodes were added by PCR amplification (12 cycles), and the cDNA libraries were purified using the Monarch PCR & DNA Cleanup Kit (NEB). High-throughput sequencing was performed on an Illumina NextSeq 550 in paired-end mode with 75 cycles per end for E. coli samples and 150 cycles per end for C. senegalense samples.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
RNA-seq data were processed using cutadapt v4.2 to remove adapter sequences, trim low-quality ends from reads, and exclude reads shorter than 18 bp.
Trimmed and filtered reads were mapped to a reference (ATCC25772 or concateinated BL21 genome (GenBank accession GCA_013166975.1) and plasmid) using bwa-mem2 v2.2.1 with default parameters. SAMtools v1.17 was used to filter uniquely mapped reads (MAPQ > 1), as well as used to sort and index the uniquely mapped reads.
Coverage tracks were generated using bamCoverage v3.5.1 with a bin size of 1, read extension to fragment size, and normalization by counts per million mapped reads (CPM) with exact scaling.
Assembly: For Cse native RNA-seq, the reference genome can be found at https://genomes.atcc.org/genomes/a91dae517b3c42e7. For all others, reference files can be found in Žedaveinytė et al. (2024) Supplementary table 3.
Supplementary files format and content: bigWig (.bw) files represent normalized files, generated using deepTools2 bamCoverage v3.5.1.
|
| |
|
| Submission date |
Mar 11, 2024 |
| Last update date |
May 06, 2024 |
| Contact name |
Samuel Henry Sternberg |
| E-mail(s) |
shsternberg@gmail.com
|
| Phone |
717-475-3658
|
| Organization name |
Columbia University
|
| Department |
Biochemistry and Molecular Biophysics
|
| Lab |
Sternberg Lab
|
| Street address |
701 W. 168th Street, HHSC 726
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10032 |
| Country |
USA |
| |
|
| Platform ID |
GPL21222 |
| Series (2) |
| GSE261343 |
Antagonistic conflict between transposon-encoded introns and guide RNAs (RNA-Seq) |
| GSE261344 |
Antagonistic conflict between transposon-encoded introns and guide RNAs |
|
| Relations |
| BioSample |
SAMN40379335 |
| SRA |
SRX23901862 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8140825_Cbo_heterologous_RNAseq.bw |
4.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
