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| Status |
Public on Dec 28, 2019 |
| Title |
Ecoli_control_diy_depletion |
| Sample type |
SRA |
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| Source name |
Bacterial liquid culture
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| Organism |
Escherichia coli |
| Characteristics |
strain: MG1655
media: M9 media supplemented with 0.1% casamino acids, 0.4% glycerol, 0.4% glucose, 2 mM MgSO4, and 0.1 mM CaCl2
rrna depletion: custom biotinylated oligos
perturbation: Negative control, 5 minutes
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| Growth protocol |
Cells were harvested at mid-log phase after outgrowth from a back-diluted overnight culture. E. coli and B. subtilis were grown at 37°C; C. crescentus was grown at 30°C.
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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 protocol. Briefly, samples with rRNA subtraction with treated with either a custom ribosome depletion technique or the Ribo-Zero kit (Illumina). Next, RNA was fragmented with RNA fragmentation reagents (Ambion). First strand cDNA synthesis was conducted using random primers and Superscript III (Invitrogen). To enable strand specificity, second strand synthesis was conducted using dUTP instead of dTTP with RNase H, E. coli DNA ligase, and E. coli DNA polymerase. Ends were repaired with T4 DNA polymerase, Klenow DNA polymerase, and T4 PNK. 3’ ends were adenylated with the Klenow fragment (3’>5’ exo-). Y-shaped adapters were ligated and 9-12 cycles of PCR were conducted with standard Illumina primers with multiplexing indexes. Amplified libraries were cleaned up with AMPure XP beads and submitted for sequencing at the MIT BioMicro Center using standard Illumina protocols.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
rRNA subtracted total RNA
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| Data processing |
Single-end reads mapped to respective genomes using bowtie2.
For the majority of analyses (all processed csv files except those ending in "density"), a single count was added for each read at the middle of the aligned region (strand, genomic_position). These counts could then be summed for genes (i.e. coding regions, rRNA) to assess rRNA depletion and quantify gene expression.
For processed csv files ending in "density", a single count was added at each aligned position in a read (e.g. a read of length 75 adds 75 total counts, 1 at each aligned position). These density maps could then be used to assess depletion across the rRNA genes.
Genome_build: NC_000913.2 (E. coli), NC_000964.3 (B. subtilis), NC_011916.1 (C. crescentus)
Supplementary_files_format_and_content: csv files record counts (see data processing) at each position in the genome. Each row corresponds to a single nt position, the two columns correspond to the positive and negative strand, respectively.
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| Submission date |
Dec 27, 2019 |
| Last update date |
Dec 30, 2019 |
| 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 |
GPL21222 |
| Series (1) |
| GSE142656 |
A simple, cost-effective, and robust method for rRNA depletion in RNA-sequencing studies |
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| Relations |
| BioSample |
SAMN13690824 |
| SRA |
SRX7449223 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4237304_190628_Ec_ctl_NextSeq_diy_depletion.csv.gz |
2.0 Mb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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