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| Status |
Public on Jan 14, 2025 |
| Title |
RNA_quant_MPRA_3 |
| Sample type |
SRA |
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| Source name |
bacterial cell
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| Organism |
Bacillus subtilis |
| Characteristics |
cell type: bacterial cell
strain: BJT200
genotype: W168 ganA::{comK kan}
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| Treatment protocol |
For all MPRA experiments, cells were grown in LB medium with an inducer (20 ng/mL anhydrotetracycline or 2% xylose) when noted. For all experiments in an inducible RNase J1 background, 2% xylose was consistently used to maintain RNase J1 expression, which was substituted for 2% glucose when RNase J1 depletion was required.
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| Growth protocol |
To collect samples, a single cryovial of a B. subtilis library was thawed, washed twice with 10 mL LB to remove glycerol, and grown overnight in 200 mL of LB at 37°C in a 2.8 L flask with vigorous shaking. These cultures were then back-diluted to an OD600 of 0.0003 in 200 mL LB with library inducer and grown at 37°C until an OD600 of 0.3, at which point samples were harvested.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from frozen cell pellets with gDNA depletion using an RNeasy Plus Mini Kit (Qiagen). gDNA was prepared from a frozen cell pellet using the Wizard Genomic DNA Purification Kit (Promega).
For RNA-derived libraries from experiments 1a, 1b, 2a, 2b, and 9 (see Table S7), 40 µg of total RNA was reverse transcribed using primer oJT442 and SuperScript III. This cDNA was then run on a 6% TBE-Urea gel, and the following size selection ranges were gel purified: 300-500 nt for full-length cggR and glnR constructs (experiments 1a, 2a, 9) and 100-250 nt for cleaved cggR constructs (experiments 1b and 2b). For the remaining experiments, cleaved RNA was isolated by size selection of RNAs ranging from 200-400 nt for tetM-cggR sites, 150-300 nt for aprE-cggR sites, and 150-350 nt for aprE-glnR sites on a 6% TBE-Urea gel (Novex). The size selected product was reverse transcribed using primer oJT442 and SuperScript III (Thermo Fisher) and size selected on 6% TBE-Urea gel, extracting approximately 120 to 200 nucleotides for cggR libraries and 100 to 200 for glnR. For all experiments, size-selected cDNA was amplified using Phusion DNA polymerase (NEB), adding a barcode and Illumina adapters with a Truseq indexing primer and an isoform-specific primer (see Table S2 for primer sequences). These isoform-specific primers were oJT441/oJT1290 for cleaved/full-length products from cggR constructs or oJT890/oJT1291 for cleaved/full-length products from glnR constructs. gDNA-derived libraries were generated using a two-step Phusion PCR protocol. An initial amplicon was performed generated through 2 cycles of PCR using from 4 µg of purified nucleic acid per 50 µL PCR reaction (reaction scaled up as needed to minimize UMI collapse in more complex libraries) with primers oJT444/445 for cggR upstream mutations, oJT1125/445 for cggR downstream mutations, and oJT891/445 for glnR downstream mutations. The untranslated cggR library required a longer amplicon to capture the upstream stop codon and was instead amplified with oJT942 and oJT445. This first PCR reaction was cleaned up with a double-sided Select-a-Size DNA Clean & Concentrator column (Zymo) and a DNA Clean & Concentrator 5 column (Zymo). A quarter of this reaction was then used for the second round of PCR, using the same primers as used for quantifying cleaved RNA abundance (oJT441 or oJT890 with our Truseq indexing primer) and size selected on an 8% TBE gel.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
mpra_3.txt
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| Data processing |
Barcodes were counted by directly parsing the single-end sequencing reads derived from both gDNA and RNA in a FASTQ format, with the barcode appearing at a fixed position within the read. As before, any variant with mutations in the constant sequence proceeding the barcode was discarded. Additionally, any two barcodes of identical sequence and unique molecular identifier were counted as a single read. The number of unique reads mapping to each variant barcode was calculated for both the RNA and gDNA-derived samples, and the ratio of RNA to gDNA-derived reads was calculated to extract the relative accumulation of processed mRNA for each variant. Barcodes can then be grouped by associated mutations, thresholded based on read count, and normalized to the median wild-type barcode’s ratio.
Assembly: N/A
Supplementary files format and content: .txt files are tab-separated tables summarizing data including counts for each detected variant, integrating information from all three datasets (barcode-to-sequence mapping, RNA quantification, and gDNA quantification).
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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) |
| GSE275081 |
A high-resolution view of RNA endonuclease cleavage in Bacillus subtilis [mpra_quant] |
| GSE275083 |
A high-resolution view of RNA endonuclease cleavage in Bacillus subtilis |
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| Relations |
| BioSample |
SAMN43228141 |
| SRA |
SRX25737681 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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