 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 06, 2024 |
| Title |
C. botulinum TnpB Input |
| Sample type |
SRA |
| |
|
| Source name |
MG1655
|
| Organism |
Escherichia coli |
| Characteristics |
cell line: MG1655
rip antibody: none
|
| Growth protocol |
E. coli str. K-12 substr. MG1655 (sSL0810) was transformed with plasmids encoding ωRNA and 3×FLAG-CboTnpB (pSL5412) or 3×FLAG-CboTnpB(D189A) (pSL5413). 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) at 100× dilution and grown until OD600 reached 0.5.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
10 mL of culture was centrifuged at 4,000 g for 10 min at 4 °C, and the supernatant was removed. The pellet was washed once with 1 mL of cold TBS (20 mM Tris-HCl (pH 7.5 at 25 °C), 150 mM NaCl), centrifuged at 10,000 g for 5 min at 4 °C, the supernatant was removed, and the resulting pellet was flash-frozen in liquid nitrogen. Pellets were stored at -80 °C. Antibodies for immunoprecipitation were conjugated to magnetic beads as follows: for each sample, 30 μL Dynabeads Protein G (Thermo Fisher Scientific) were washed 3× in 1 mL RIP lysis buffer (20 mM Tris-HCl (pH 7.5 at 25 °C), 150 mM KCl, 1 mM MgCl2, 0.2% Triton X-100), resuspended in 1 mL RIP lysis buffer, combined with 10 μL anti-FLAG M2 antibody (Sigma-Aldrich, F3165), and rotated for >3 hours at 4 °C. Antibody-bead complexes were washed 3× to remove unconjugated antibodies, and resuspended in 30 μL RIP lysis buffer per sample. To generate cell lysates, flash-frozen pellets were first resuspended in 1.2 mL RIP lysis buffer supplemented with cOmplete Protease Inhibitor Cocktail (Roche) and SUPERase•In RNase Inhibitor (Thermo Fisher Scientific). Cells were then sonicated for 1.5 min total (2 s ON, 5 s OFF) at 20% amplitude. To clear cell debris and insoluble material, lysates were centrifuged for 15 min at 4 °C at 21,000 g, and the supernatant was transferred to a new tube. At this point, a small volume of each sample (24 μL, or 2%) was set aside as the “input” starting material and stored at -80 °C.For immunoprecipitation, each sample was combined with 30 μL antibody-bead complex and rotated overnight at 4 °C. The next day, each sample was washed 3× with ice-cold RIP wash buffer (20 mM Tris-HCl (pH 7.5 at 25 °C), 150 mM KCl, 1 mM MgCl2). After the last wash, beads were resuspended in 1 mL TRIzol (Thermo Fisher Scientific) and incubated at RT for 5 min to allow separation of RNA from the beads. A magnetic rack was used to isolate the supernatant, which was transferred to a new tube and combined with 200 μL chloroform. Each sample was mixed vigorously by inversion, incubated at RT for 3 min, and centrifuged for 15 min at 4 °C at 12,000 g. RNA was isolated from the upper aqueous phase using the RNA Clean & Concentrator-5 kit (Zymo Research) and eluted in 15 μL RNase-free water. RNA from input samples was isolated in the same manner using TRIzol and column purification.
For RIP-seq library preparation (input and RIP eluates), 6 μL of RNA was diluted in FastAP Buffer (Thermo Fisher Scientific) supplemented with SUPERase•In RNase Inhibitor (Thermo Fisher Scientific) to a total volume of 18 μL, and fragmented by heating to 92 °C for 1.5 min. Each sample was treated with 2 μL TURBO DNase (Thermo Fisher Scientific) for 30 min at 37 °C and column-purified using the RNA Clean & Concentrator-5 kit (Zymo Research), eluting in 12.5 μL RNase-free water. RNA concentration was quantified using the DeNovix RNA Assay. Illumina sequencing libraries were prepared using the NEBNext Small RNA Library Prep kit, and libraries were sequenced on an Illumina NextSeq 500 in paired-end mode with 75 cycles per end.
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| 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 (concatenated MG1655 genome (GenBank accession AKVX01000001.1) 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: 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) |
| GSE261342 |
Antagonistic conflict between transposon-encoded introns and guide RNAs (RIP-Seq) |
| GSE261344 |
Antagonistic conflict between transposon-encoded introns and guide RNAs |
|
| Relations |
| BioSample |
SAMN40379330 |
| SRA |
SRX23901881 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8140822_G02_unique.bw |
2.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
