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| Status |
Public on Jan 14, 2025 |
| Title |
BC_mapping_MPRA_6 |
| Sample type |
SRA |
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| Source name |
bacterial cell
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| Organism |
Bacillus subtilis |
| Characteristics |
strain: GLB115
cell type: bacterial cell
genotype: 168 (trpC2)
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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 |
genomic DNA |
| Extraction protocol |
gDNA was prepared from a frozen cell pellet using the Wizard Genomic DNA Purification Kit (Promega).
gDNA-derived libraries were generated using a two-step Phusion PCR protocol. An initial amplicon was 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 primers oJT446/447. This product was size selected on an 8% TBE gel and sequenced.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Data processing |
Barcode quantification and variant sequence identification was handled using custom scripts written in Python. To determine the variant sequence associated with each barcode, the paired end reads from the gDNA-derived libraries were directly parsed as FASTQ files, with one read capturing the barcode and the other the variable region of the construct. Variants with at least one mutation in the constant region proceeding the barcode were discarded. To avoid rare mutations or sequencing errors confounding our variant sequence mapping, variant sequence assignment required at least three reads per barcode. If, among these reads, the second-most frequent sequence was at least 25% as frequent as the top sequence, the barcode was discarded entirely.
Assembly: N/A
Supplementary files format and content: .txt files are tab-separated tables containing information and 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 |
GPL21373 |
| Series (2) |
| GSE275080 |
A high-resolution view of RNA endonuclease cleavage in Bacillus subtilis [mpra_bc_mapping] |
| GSE275083 |
A high-resolution view of RNA endonuclease cleavage in Bacillus subtilis |
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| Relations |
| BioSample |
SAMN43228104 |
| SRA |
SRX25737654 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8465414_mpra_6.txt.gz |
980.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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