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| Status |
Public on Aug 11, 2024 |
| Title |
KpnDRT2_WT_rep3_infected_input |
| Sample type |
SRA |
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| Source name |
MG1655
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| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
strain: MG1655
cdip antibody: none
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| Treatment protocol |
For experiments +/- phage infection, 40 mL cultures were split in half, and phage T5 was added to one half at a multiplicity of infection (MOI) of 5, which was calculated as the ratio of phage plaque forming units (PFU) to bacterial colony forming units (CFU). Uninfected and infected cultures were grown for 1 hr at 37 °C. For experiments without phage infection, 20 mL cultures were grown to OD600 of 0.5 and directly harvested.
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| Growth protocol |
E. coli str. K-12 substr. MG1655 (sSL0810) was transformed with plasmids encoding C-terminally 3×FLAG-tagged Retron-Eco1 or N-terminally 3×FLAG-tagged KpnDRT2 (WT, RT mutant, or ncRNA mutant), as well as their native flanking sequences. Individual colonies were inoculated in liquid LB with chloramphenicol and grown at 37 °C to OD600 of 0.5.
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| Extracted molecule |
other |
| Extraction protocol |
Cells were harvested by centrifugation at 4,000 x g for 10 min at 4 °C, and the supernatant was removed. The pellet was washed with 5 mL of cold TBS (20 mM Tris-HCl, pH 7.5 at 25 °C, 150 mM NaCl) and spun down again as before. The supernatant was removed, and the pellet was washed with 1 mL of cold TBS before centrifugation at 10,000 x g for 5 min at 4 °C. The supernatant was removed, and the pellet was flash-frozen in liquid nitrogen and stored at -80 °C. Antibodies for immunoprecipitation were conjugated to magnetic beads as follows: for each sample, 60 μL Dynabeads Protein G (Thermo Fisher Scientific) were washed 3× in 1 mL IP 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 IP lysis buffer, combined with 20 μL anti-FLAG M2 antibody (Sigma-Aldrich, F3165), and rotated for > 3 hours at 4 °C. Antibody-bead complexes were washed 2× to remove unconjugated antibodies and resuspended in 60 μL of IP lysis buffer per sample. Flash-frozen pellets were thawed on ice and resuspended in 1.2 mL IP lysis buffer supplemented with 1× cOmplete Protease Inhibitor Cocktail (Roche) and 0.1 U µL−1 SUPERase•In RNase Inhibitor (Thermo Fisher Scientific). To lyse cells, samples were sonicated using a 1/8″ sonicator probe for 1.5 min total (2 s ON, 5 s OFF) at 20% amplitude. To clear cell debris and insoluble material, lysates were centrifuged at 21,000 x g for 15 min at 4 °C, and 1 mL supernatant was transferred to a new tube. At this point, 10 µL of each sample were set aside as “input” starting material and stored at -80 °C. For immunoprecipitation, each sample was combined with 60 μL antibody-bead complex and rotated overnight at 4 °C. The next day, each sample was washed 3× with 1 mL ice-cold IP wash buffer (20 mM Tris-HCl, pH 7.5 at 25 °C, 150 mM KCl, 1 mM MgCl2), using a magnetic rack to immobilize the beads in between each wash. For cDIP elution, the supernatant was removed, and beads were resuspended in 90 µL IP wash buffer and treated with 5 µg RNase A (Thermo Fisher Scientific) for 30 min at 37 °C. Input samples were adjusted to 90 µL with IP wash buffer and treated with RNase A in parallel. SDS was added to IP and input samples to a final concentration of 1%, and samples were treated with 25 µg Proteinase K (Thermo Fisher Scientific) for 30 min at 55 °C. Beads were immobilized using a magnetic rack, and the supernatant containing eluted DNA was transferred to a new tube. DNA was isolated using the Monarch PCR and DNA Cleanup kit (NEB), following the Oligonucleotide Cleanup protocol and eluting in 15 µL DNase-free water. For Retron-Eco1 samples, DNA was treated with DBR1 (Origene) in reactions containing 2 µL DNA, 0.5 µL DBR1, 1× rCutSmart in 10 µL total volume, in order to cleave the 2′-5′ phosphodiester linkage between msRNA and msDNA. Reactions were cleaned up using the Monarch PCR and DNA Cleanup kit (NEB), with elution in 15 µL DNase-free water. Purified DNA was stored at -80 °C before proceeding to library preparation.
For cDIP-seq library preparation, 2 µL of each input sample and 10 µL of each IP eluate were diluted to 15 µL with DNase-free water. Samples were denatured by heating at 95 °C for 2 min, and then immediately placed on ice. Ligation of Illumina adapters and conversion of ssDNA to dsDNA were performed using the xGen ssDNA & Low-Input DNA Library Prep Kit (IDT), and libraries were sequenced on an Illumina NextSeq 500 in paired-end mode with 150 cycles per end.
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| Library strategy |
OTHER |
| Library source |
other |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
cDIP-seq and corresponding input datasets were processed using cutadapt (v4.2) to remove Illumina adapter sequences, trim low-quality ends from reads, and filter out reads shorter than 15 bp. Reads were mapped to combined reference files containing the MG1655 genome (NC_000913.3) and relevant plasmid sequence, as well as the T5 genome (NC_005859.1) for +/- infection experiments, using bwa-mem2 (v2.2.1) with default parameters. SAMtools (v1.17) was used to sort and index alignments. Coverage tracks were generated using bamCoverage (v3.5.1) with a bin size of 1, separation of top and bottom strand alignments, and scaling of coverage according to sequencing depth (based on the total number of reads passing initial trimming and length filtering).
Assembly: Genome reference sequences can be found in the NCBI database via the accession numbers NC_000913.3 (E. coli MG1655) and NC_005859.1 (phage T5). Plasmid reference sequences can be found in Tang et al. (2024) Table S3.
Supplementary files format and content: bigWig (.bw) files represent normalized read coverage files, separated by strand.
Library strategy: cDIP-seq
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| Submission date |
Jun 18, 2024 |
| Last update date |
Aug 12, 2024 |
| Contact name |
Samuel Henry Sternberg |
| E-mail(s) |
shsternberg@gmail.com
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| Phone |
717-475-3658
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| Organization name |
Columbia University
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| Department |
Biochemistry and Molecular Biophysics
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| Lab |
Sternberg Lab
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| Street address |
701 W. 168th Street, HHSC 726
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10032 |
| Country |
USA |
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| Platform ID |
GPL21117 |
| Series (2) |
| GSE270160 |
De novo gene synthesis by an antiviral reverse transcriptase [cDIP-seq] |
| GSE270164 |
De novo gene synthesis by an antiviral reverse transcriptase |
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| Relations |
| SRA |
SRX24966651 |
| BioSample |
SAMN41895902 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8335740_ST138_09i_D_fwd.bw |
18.0 Mb |
(ftp)(http) |
BW |
| GSM8335740_ST138_09i_D_rev.bw |
17.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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