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| Status |
Public on Jul 13, 2021 |
| Title |
rz_5m-yafo2 |
| Sample type |
SRA |
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| Source name |
bacterial liquid culture
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| Organism |
Escherichia coli |
| Characteristics |
strain: MG1655 pBAD30-yafo
induction time: 5 minutes
rrna depletion: Yes
trna size selection: No
fragmentation time: 0 minutes
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| Growth protocol |
Escherichia coli was grown in M9 (10x stock made with 64 g/L Na2HPO4-7H2O, 15 g/L KH2PO4, 2.5 g/L NaCl, 5.0 g/L NH4Cl) medium supplemented with 0.1% casamino acids, 0.4% glycerol, 2 mM MgSO4, and 0.1 mM CaCl2. Single colonies on an LB + 0.4% glucose plate were picked and grown overnight in M9 media + 0.4% glucose on a roller drum then grown at 37°C on a Synergy H1 plate reader (BioTek) in 24-well plates using double orbital rotation at 307 CPM for the course of the experiment. Overnight cultures grown with 0.4% glucose were back-diluted to 0.02 OD600 in 1.5 mL of M9 media with added glucose and grown to OD 0.2 – 0.35 in 24-well plates. Cultures were pelleted by spinning 5 minutes on a benchtop centrifuge at 4000 G. Pellets were washed once with M9 media lacking glucose, respun, and resuspended in M9 media lacking glucose. On a fresh 24-well plate, individual cultures were back-diluted to 0.1 and returned to the plate reader. After 30 minutes of growth, toxin was induced by adding arabinose to 0.2% and cells were returned to the plate reader. After induction for either 5 or 30 minutes, 1 mL of the culture was mixed with 110 uL of stop solution (95% ethanol and 5% phenol), spun for 30 seconds at 13k RPM to pellet cells, and flash frozen in liquid nitrogen.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using Trizol (Invitrogen) and Direct-zol RNA MiniPrep kit (Zymo).
See publication for complete details. For samples with rRNA depletion, rRNA was removed by hybridization to custom biotinylated oligos. Next, if samples were fragmented, RNA was mixed with reverse transcription buffer and heated to 95°C for 3 minutes before placing on ice. All samples were then reverse transcribed using reverse transcription primers with a 5’-overhang with a PCR amplification handle and 1 of 24 inline barcodes. As cDNA now contained a barcode, samples were pooled and those with a tRNA size selection step were loaded onto a denaturing TBE urea gel and the tRNA region of the gel was extracted for cDNA purification. For all other samples, pools were cleaned up with a modified AMPure XP bead protocol to remove leftover primers. Next, all samples were subjected to a second round of reverse transcription with a template switching oligo to add a second PCR amplification handle to the 3’-end of the cDNA. After a bead cleanup, samples were PCR amplified with primers adding the i5 and i7 handles for Illumina sequencing. PCR reactions were size selected with a 2-sided bead cut and sequenced using a custom read 1 primer on a NovaSeq instrument. The combination of the i7 index and the inline read 2 barcode were used to separate out biological samples into their own fastq files using cutadapt.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
rRNA depleted total RNA
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| Data processing |
The inline barcode was clipped from read 2 and used to separate reads into their source samples using cutadapt (v3.2). Adapter sequences were also trimmed at this stage – the provided fastq files are both demultiplexed and trimmed.
Reads were mapped to the MG1655 genome using bowtie2 (v2.4.1) using the --very-sensitive argument set.
For processed files ending in “5_prime_ends”: for each proper pair, 1 count was added to the 5’-end of the fragment (corresponding to a free RNA 5’-end). For processed files ending in “density”: for each proper pair, 1 count was added to every position aligning to and between the two reads.
Genome_build: U00096.3
Supplementary_files_format_and_content: Processed files are gzipped csv files. The first column corresponds to the forward strand and the second two the reverse strand. Each row corresponds to the counts at a genomic position from 1 to len(genome). Counts are defined in the data processing steps. Integer counts are raw and not normalized to library depth.
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| Submission date |
Jul 06, 2021 |
| Last update date |
Jul 14, 2021 |
| Contact name |
Peter Culviner |
| Organization name |
MIT
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| Department |
Biology
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| Lab |
Laub
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| Street address |
31 Ames St
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
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| Platform ID |
GPL25368 |
| Series (1) |
| GSE179607 |
Global analysis of the specificities and targets of endoribonucleases from E. coli toxin-antitoxin systems |
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| Relations |
| BioSample |
SAMN20085788 |
| SRA |
SRX11365038 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5421569_rz_5m-yafo2_5_prime_ends.csv.gz |
493.5 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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