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Status |
Public on Jan 14, 2025 |
Title |
Phage_cured_rnjA_rendseq_pooled |
Sample type |
SRA |
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Source name |
bacterial cell
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Organism |
Bacillus subtilis |
Characteristics |
cell type: bacterial cell strain: BG880 genotype: W168 {delta}Skin {delta}SP{beta}::PIID-sspB kan rnjA::spc
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Growth protocol |
All Rend-seq sequencing experiments were performed using cells grown in 2xYT, supplemented with 2% xylose or glucose for RNase J1 induction or depletion as necessary. Except when we sought to deplete RNase J1, strains with an inducible RNase J1 allele were consistently grown in the presence of 2% xylose. For all sequencing experiments, overnight cultures in 2xYT were back-diluted to an OD600 of 0.0002 in 20 mL prewarmed 2xYT and grown to an OD600 of 0.2, at which point they were harvested. For any strain with an RNase J1 knockout, cultures were vortexed prior to and following inoculation to minimize clumping. To achieve RNase J1 depletion, 10 times the required volume of overnight culture was collected, washed twice with 1 mL pre-warmed 2xYT, and resuspended in 1 mL 2xYT. 100 µL of this resuspension was used to inoculate the final culture. Samples were harvested for sequencing by adding 7 mL of culture to 7 mL of ice cold methanol, pelleting, decanting, and flash freezing with liquid nitrogen. Cell pellets were stored at -80°C prior to RNA extraction.
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Extracted molecule |
total RNA |
Extraction protocol |
Prior to RNA extraction, cell pellets were washed with 10-15 mL ice cold 10 mM Tris pH 7.0 to remove contaminating nucleic acids that precipitate from 2xYT in methanol. For Rend-seq, RNA was extracted from cell pellets using either an RNeasy Mini Kit (Qiagen) with on-column DNase treatment, an RNeasy Plus Mini Kit (Qiagen) or through RNAsnap extraction (Stead et al., 2012) followed by Turbo DNase treatment. Following RNA extraction and gDNA depletion, 20 µg of purified RNA was treated with MICROBExpress Bacterial mRNA Enrichment Kit (ThermoFisher) per the manufacturer’s instructions and precipitated with isopropanol. Libraries were constructed as per the Rend-seq protocol described in Lalanne et al., Cell 2018.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NextSeq 500 |
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Data processing |
Adapter or poly(A) sequence was trimmed from end sequencing and Rend-seq libraries using CutAdapt (Martin, 2011) with parameters “-a 'CTGTAGGCACCATCAAT' -m 15”. Trimmed reads were then mapped to the S. cerevisiae genome (SacCer3) using bowtie v1.1.2 with parameters “-v 1 -k 1 –best –un” with retained unmapped reads subsequently mapped to the B. subtilis genome (NC_000964.3) with the same parameters (Langmead et al., 2009). Using custom Python scripts, we converted the bowtie output to wig files, attributing signal from reads with a mismatch at their first position to the adjacent nucleotide to address mismatches due to non-templated addition during reverse transcription. Assembly: B. subtilis NC000964.3 Supplementary files format and content: Wiggle file with two columns: first column contains chromosome positions and second column contains the number of reads its 5' or 3' end mapped to the position.
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Submission date |
Aug 16, 2024 |
Last update date |
Jan 14, 2025 |
Contact name |
James Christopher Taggart |
E-mail(s) |
james_taggart@hms.harvard.edu
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Organization name |
Harvard Medical School
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Department |
Systems Biology
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Lab |
Allon Klein
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Street address |
200 Longwood Avenue
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City |
Boston |
State/province |
MA |
ZIP/Postal code |
02115 |
Country |
USA |
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Platform ID |
GPL24109 |
Series (2) |
GSE275076 |
A high-resolution view of RNA endonuclease cleavage in Bacillus subtilis [rend_seq] |
GSE275083 |
A high-resolution view of RNA endonuclease cleavage in Bacillus subtilis |
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Relations |
BioSample |
SAMN43227652 |
SRA |
SRX25737204 |
Supplementary file |
Size |
Download |
File type/resource |
GSM8465250_PC_J1_Rend_pooled_trimmed_filtered-3-f.wig.gz |
2.3 Mb |
(ftp)(http) |
WIG |
GSM8465250_PC_J1_Rend_pooled_trimmed_filtered-3-r.wig.gz |
2.1 Mb |
(ftp)(http) |
WIG |
GSM8465250_PC_J1_Rend_pooled_trimmed_filtered-5-f.wig.gz |
2.2 Mb |
(ftp)(http) |
WIG |
GSM8465250_PC_J1_Rend_pooled_trimmed_filtered-5-r.wig.gz |
2.1 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
Raw data are available in SRA |
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