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| Status |
Public on Mar 16, 2021 |
| Title |
pCF10_P23IGR_pCIE_U |
| Sample type |
SRA |
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| Source name |
bacterial cellular RNA from pulldown
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| Organism |
Enterococcus faecalis |
| Characteristics |
strain: OG1RF pCF10
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| Treatment protocol |
cCF10 was added at a final concentration of 20 ng/mL and cultures were grown to an OD600 of 0.3. Cells were pelleted by centrifugation at 16,000 x g at 4 °C for 5 min, washed once with 10 mL TBS buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl), and resuspended in 1 mL TBS and transferred to a 1.5 mL eppendorf tube. Cells were pelleted by centrifugation at 16,000 x g at 4 °C for 5 min, the supernatant was removed, and the cell pellet was flash frozen in a dry ice-ethanol slurry and stored at -80 °C.
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| Growth protocol |
E. faecalis strains were grown overnight in BHI (5 mL), and then diluted to an OD600 of ~0.03 in 200 mL BHI and grown without shaking to an OD600 of 0.1.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cell lysates were prepared by suspending cells in 500 µL of TBS buffer with 5 µL of HALT protease inhibitor cocktail and 2 µL of superase. Cells were added to a pre-chilled tube containing 500 µL of glass beads (0.1 mm) and disrupted by repeated cycles of maceration for 40 sec at 400 rpm with intervals on ice. TBS buffer (500 µL) was added and the slurry was vortexed for 30 sec, after which cell fragments and other material were pelleted by centrifugation 12,000 x g for 30 min at 4 °C. The supernatant was transferred to a pre-chilled tube, the volume was increased to 1 mL with cold TBS buffer, and 5 µL of superase inhibitor was added. An aliquot representing the input fraction was removed, and the remaining material was subjected to FLAG pull-downs by addition of 75 µL of anti-FLAG magnetic bead slurry to a 2.0 mL dolphin tube (Costar 3213). The mixture was then placed on a nutator (rocker) for 2.0 h at 4 °C, after which time the resin was pelleted by centrifugation at 4,000 x g for 30 sec at 4 °C. The supernatant was removed, and the resin washed 3 x with 1.5 mL of TBS buffer, with incubations on a nutator for 1 min and then subjected to magnetic bead pull-downs at 4 °C. Material bound to the FLAG beads was eluted by addition of 15 µL 5 µg/µL 3xFLAG peptide solution and 250 µL of 3xFLAG elution buffer. Ribosomal RNA was depleted using a MicrobeExpress kit. All RNA samples were treated with Turbo DNase (ThermoFisher, rigorous method described by the manufacturer) and submitted to the University of Minnesota Genomics Center for Illumina library prep.
Pull-down samples were processed using the Illumina TruSeq stranded mRNA preparation kit. To retain small RNAs, size selection was not performed between library preparation and sequencing for pull-down samples. All samples were sequenced as paired-end reads on an Illumina HiSeq 2500 in high output mode (125 bp read length for whole-cell samples and 50 bp read length for pull-down samples). Sequencing files were trimmed to remove contaminating adapter sequences and low-quality bases using Trimmomatic. Reads were mapped to the pCF10
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Sequenced reads were trimmed to remove adapter sequences and low-quality bases using the sliding-window option in Trimmomatic
RPKM and Expression values were calculated using Rockhopper
Genome_build: NC_006827
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| Submission date |
Mar 15, 2021 |
| Last update date |
Jul 16, 2021 |
| Contact name |
Julia Willett |
| E-mail(s) |
jwillett@umn.edu
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| Organization name |
University of Minnesota
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| Street address |
689 23rd Avenue SE, 4-149 Micro Research Facility
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| City |
Minneapolis |
| State/province |
MN |
| ZIP/Postal code |
55455 |
| Country |
USA |
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| Platform ID |
GPL23172 |
| Series (1) |
| GSE168958 |
Pulldown of RNA using PrgU-FLAG from Enterococcus faecalis OG1RF pCF10 |
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| Relations |
| BioSample |
SAMN18314660 |
| SRA |
SRX10347089 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5172774_Galaxy7-_JW_A9_S9_R1_001_Galaxy8-_JW_A9_S9_R2_001_NC_006827_v1_m15_aT_d500_l33_rf_cF.minus.wig.gz |
8.0 Kb |
(ftp)(http) |
WIG |
| GSM5172774_Galaxy7-_JW_A9_S9_R1_001_Galaxy8-_JW_A9_S9_R2_001_NC_006827_v1_m15_aT_d500_l33_rf_cF.plus.wig.gz |
5.5 Kb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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