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| Status |
Public on Jan 27, 2016 |
| Title |
BG578 PARE |
| Sample type |
SRA |
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| Source name |
BG578_PARE_RNA-seq
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| Organism |
Bacillus subtilis |
| Characteristics |
strain: BG578
genotype (on ribonucleases): Pspac-rnjA ery
growth condition: 37°C LB medium to mid-log phase
molecule subtype: Total RNA with rRNA removed
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| Growth protocol |
The cells were cultured in LB medium at 37°C until mid-log phase.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with hot phenol method, and ribosomal RNA was removed with Ribo-Zero rRNA Removal Kit for bacteria
For regular RNA-seq, Libraries were constructed according to the standard Illumina RNA-seq protocol. For PARE, equal amounts of samples were used to construct the PARE libraries using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (Set 1). The manufacturer’s protocol was modified so that 5ʹ monophosphorylated ends that result from endonuclease cleavage or pyrophosphohydrolase activity were preferentially selected. Briefly, after 3ʹ SR Adaptor ligation, reverse transcriptase (RT) primer hybridization, and 5ʹ SR Adaptor ligation – performed as per the manufacturer’s protocol – the reaction mixture volume was brought to 100 μl and samples were cleaned up with the RNeasy MinElute Cleanup Kit (Qiagen). 12 μl of RNA was recovered following the manufacturer’s protocol. The RNA was fragmented using the NEBNext® Magnesium RNA Fragmentation Module at 94°C for 4 minutes, and 2 μl of stop solution was added. After a second RNeasy MinElute cleanup, half of the RNA (6 μl) was again ligated to the 3ʹ SR Adaptor. Thus, after magnesium fragmentation only RNAs that retained their 5ʹ SR Adaptor (and original 5’ monophosphate) would undergo subsequent RT and PCR amplification. After addition of the RT primer, the reaction volume was adjusted to 30 μl and RT/PCR amplification reactions were performed as per the manufacturer’s protocol, using barcoded primers. One tenth of the PCR reaction was run on a 1.5% agarose gel to visualize the library, which consisted of cDNA ranging from 50 to ~500 nts. 20 μl of the cDNA library were prepared for Illumina sequencing using a 1.5X volume of Agencourt AMPure XP Beads (Beckman Coulter), according to the manufacturer’s instructions. The procedure was repeated twice, and the sample was eluted in a final volume of 20 µl.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
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| Description |
PARE
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| Data processing |
Base-calling was performed by Illumina CASAVA software.
Reads from fastq files were aligned to Bacillus subtilis genome by Bowtie 2 with --local option.
SAMtools and BEDtools were used to count reads mapped to specific genes for regular RNA-seq. Gene annotation reference was obtained from NCBI database. Homemade Python scripts were used to count read coverage at each base and to pick the peaks with >100 reads per position and >25nt in length for PARE experiments.
For regular RNA-seq, homemade Python script was used to normalize and compare the read abundance (reads/base) of each transcript as described in a previous study (Liu, B., et al. 2014 PMID: 25099370).
For PARE, homemade Python script was used to calculate an RR ratio for each peak found in BG880, which is the ratio of read coverage in the specific peak region in BG880 and BG879 normalized by the expression data of this region from regular RNA-seq (peak_100_RR.txt)
Genome_build: NC_000964.3
Supplementary_files_format_and_content: Tab-delimited txt files contain peaks (peaks_10_BG*.txt) or read abundance for genes (expression.txt)
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| Submission date |
Jan 26, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
David H Bechhofer |
| E-mail(s) |
david.bechhofer@mssm.edu
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| Phone |
212-241-5628
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| Organization name |
Icahn School of Medicine at Mount Sinai
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| Department |
Department of Pharmacology and Systems Therapeutics
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| Street address |
One Gustave L. Levy Place, Box 1603
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10029 |
| Country |
USA |
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| Platform ID |
GPL21373 |
| Series (1) |
| GSE77217 |
Mapping of internal monophosphate 5’ ends of Bacillus subtilis messenger RNAs and ribosomal RNAs in wild-type and ribonuclease-mutant strains |
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| Relations |
| BioSample |
SAMN04440604 |
| SRA |
SRX1547389 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2046123_peaks_100_BG578.txt.gz |
25.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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