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Sample GSM8172008 Query DataSets for GSM8172008
Status Public on May 08, 2024
Title Caulobacter_early_stationary_R3
Sample type SRA
 
Source name Root343
Organism Caulobacter sp. Root343
Characteristics strain: Root343
phase: early stationary
Extracted molecule total RNA
Extraction protocol E. coli expressing the Bil system under control of a tetracycline-inducible promoter was grown to mid-log phase with 25 ng/ml aTc as inducer. Caulobacter sp. Root343 was grown to mid-log, late-log or early stationary phase. Samples corresponding to an OD600 of 2 were taken and immediately mixed with ¼ volume of stop mix (95% ethanol, 5% saturated phenol) and frozen in liquid nitrogen. The samples were then thawed on iced followed by centrifugation at 4,000 g and 4°C for 10 min. The supernatant was discarded and the cell pellets resuspended in 1 ml of TRIzol (Thermo Scientific) followed by RNA extraction according to the manufacturer’s protocol. The resulting RNA pellet was dissolved in 40 µl of water. To get rid of contaminating DNA, 0.5 µl of water, 5 µl DNase I buffer with MgCl2 (Thermo Scientific), 0.5 µl of RNase inhibitor (Thermo Scientific) and 4 µl DNase I (Thermo Scientific) was added and the mixture incubated at 37°C for 30 min. The DNase-digested RNA was subjected to acidic phenol-chloroform extraction and finally dissolved in 30 µl of water.
Extracted RNA was depleted of ribosomal RNA using a commercial rRNA depletion kit for mixed bacterial samples (Lexogen, RiboCop META, #125). The depleted RNA samples were fragmented using ultrasound (2 pulses of 30 s at 4°C). Then, an oligonucleotide adapter was ligated to the 3' end of the RNA molecules. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase with the 3’ adapter as primer. After purification, the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified using a high-fidelity DNA polymerase and the barcoded TruSeq-libraries were pooled in approximately equimolar amounts. Sequencing of pooled libraries, spiked with PhiX control library, was performed at a minimum of 10 million reads per sample in single-ended mode with 100 cycles on the NextSeq 2000 platform (Illumina).
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model NextSeq 2000
 
Description Caulobacter_TPM_counts.xlsx
Data processing Demultiplexing using bcl-convert v4.2.4
Trimming using Trim Galore v0.6.7 with default parameters
Mapping using BBMap v39.06 with default parameters
Read counting using featureCounts v2.0.3 with -s 1
Assembly: GCF_001425745.1, NC_000913.3
Supplementary files format and content: Excel file containing TPM values for each sample
 
Submission date Mar 27, 2024
Last update date May 08, 2024
Contact name Jens Hör
Organization name Weizmann Institute for Science
Lab Rotem Sorek
Street address 234 Herzl Street
City Rehovot
ZIP/Postal code 7610001
Country Israel
 
Platform ID GPL34341
Series (1)
GSE262579 Bacteria conjugate ubiquitin-like proteins to interfere with phage assembly
Relations
BioSample SAMN40627648
SRA SRX24073349

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA

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