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Status |
Public on Jan 14, 2025 |
Title |
rnjA_depletion_rny_5end_seq_pooled |
Sample type |
SRA |
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Source name |
CCB760
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Organism |
Bacillus subtilis |
Characteristics |
cell type: bacterial cell strain: CCB760 genotype: W168 Pspac-rnjA ery rny::spc
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Growth protocol |
All 5end-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 all 5′ end sequencing samples, RNA was extracted using the RNAsnap protocol and Turbo DNase treated as described for the Rend-seq and 3′ end sequencing experiments. rRNA was depleted from 10 µg of purified RNA with the MICROBExpress Bacterial mRNA Enrichment Kit, per manufacturer’s instructions, precipitated, and resuspended in 10 mM Tris pH 7.0. 600 ng rRNA depleted RNA was then ligated to 260 pmol of an RNA adapter for 18 hours at 22 °C using T4 RNA Ligase 1. Following ligation, RNA was fragmented for 2 minutes using RNA fragmentation reagents (ThermoFisher) and fragments of either size 41-71 or 60-90 were isolated through denaturing polyacrylamide gel extraction. RNA fragments were dephosphorylated, polyadenylated, and reverse transcribed following the BaM-seq multiplexed RNA sequencing protocol (Johnson et al., 2023). To add Illumina adapters, pooled cDNA was amplified between 5-10 cycles with primers oDP128 and oDP161 (Johnson et al., 2023) using Q5 DNA polymerase and purified by polyacrylamide gel electrophoresis.
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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 |
poly(A) sequence was trimmed from 5end sequencing libraries using CutAdapt (Martin, 2011) with parameters “-a A{15} -m 15 -u 8 --rename='{id} UMI={cut_prefix}’”. 5′ end sequencing reads were collapsed using the 8 bp unique molecular identifier (UMI) at the start of each read using custom Python scripts, requiring identical UMI sequences and read sequences within a hamming distance of 1 to be considered duplicates. 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. 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 with a 5' 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) |
GSE275074 |
A high-resolution view of RNA endonuclease cleavage in Bacillus subtilis [fiveprime_end_seq] |
GSE275083 |
A high-resolution view of RNA endonuclease cleavage in Bacillus subtilis |
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Relations |
BioSample |
SAMN43227502 |
SRA |
SRX25737041 |
Supplementary file |
Size |
Download |
File type/resource |
GSM8465230_indJ1_rny_5end_pooled_trimmed_filteredUMIcollapse-5-f.wig.gz |
1.7 Mb |
(ftp)(http) |
WIG |
GSM8465230_indJ1_rny_5end_pooled_trimmed_filteredUMIcollapse-5-r.wig.gz |
1.6 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
Raw data are available in SRA |
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