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| Status |
Public on May 20, 2019 |
| Title |
DK5955_B RibosomeProfiling |
| Sample type |
SRA |
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| Source name |
batch culture
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| Organism |
Bacillus subtilis |
| Characteristics |
tissue: batch culture
genotype: comIQ12 <delta>efp nusGN21S
growth medium: Lysogeny Broth
growth temperature: 37°C
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| Treatment protocol |
Cells were lysed in 650 µL lysis buffer (10 mM MgCl2, 100 mM NH4Cl, 5 mM CaCl2, 20 mM Tris pH 8.0, 0.1% NP-40, 0.4% Triton X-100, 0.1 units/µL RNase free DNase I (Invitrogen AM2222), 0.5 units/μL Superase-In (Invitrogen AM2696)) using a Spex 6875 freezer mill set to 10 cycles of 2 min runs at 15 cps separated by 2 min rests.
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| Growth protocol |
Cells were grown shaking in 300 mL lysogeny broth to an OD600 of 0.3-0.4, isolated by rapid filtration, and flash frozen in liquid nitrogen.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Following lysis, 25 A260 units of lysate were digested with 1500 units of S7 micrococcal nuclease (Roche 10107921001) for 1 hr at room temp after which the reaction was quenched by the addition of EGTA to a final concentration of 6 mM. The digested lysate was then applied to a 10%-50% sucrose gradient and centrifuged in a Ti-40 rotor at 35,000 rpm for 2.5 hrs at 4°C. 700 µL of fractions containing 70S ribosomes were denatured in 1% SDS and extracted once with an equal volume of hot acid phenol, once with an equal volume of room temp acid phenol, and RNA was precipitated with isopropanol. The precipitant was resuspended in 12 µL H2O and 25 µg RNA was mixed with 2X loading dye (10 mM EDTA, 30 μg/mL bromophenol blue, and 98% formamide) and resolved on a 15% polyacrylamide TBE Urea gen run at 250 V for 40 min. After staining the gel in SYBR Gold (Sigma S11494) for 3 min, products between ~15 and 40 bp were excised and subsequently gel extracted.
RNA 3' ends were dephosphorylated with T4 poly-nucleotide kinase (Lucigen). Dephosporylated RNA was subsequently ligated to Linker 1 (IDT /5rApp/CTGTAGGCACCATCAAT/3ddC/) with T4 RNA ligase 2, truncated (NEB) and products between 30-100 bp were gel extracted. Isolated RNA was then reverse transcribed using Superscript III (Invitrogen) with the reverse transcription primer (IDT 5′-(Phos)-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGC-(SpC18)-CACTCA-(SpC18)-TTCAGACGTGTGCTCTTCCGATCTATTGATGGTGC CTACAG-3). RNA was subsequently hydrolyzed by the addition of 2.2 µL 1N NaOH and incubation at 98C for 20 min. cDNA products were isolated by gel extraction and circularized using CircLigase (Epicentre). Circularized products were used as a template for PCR reactions using Phusion Polymerase (NEB M0530S) with forward library primer (IDT 5′-AATGATACGGCGACCACCGAGATCTACAC-3′) and Indexed reverse library primer (IDT 5′-CAAGCAGAAGACGGCATACGAGATNNNNNNNNGACTGGAGTTCAGACGTGTGCTCTTCCG-3′) where NNNNNNNN represents the barcode sequence unique to each library. After 6-10 cycles, two PCR reactions per sample with no apparent duplexed products after resolution of 2 μL on an 8% polyacrylamide TBE urea gel were pooled and DNA was purified with a QIAquick kit (Qiagen) and eluted in 20 µL H2O.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Ribosome protected mRNA fragments
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| Data processing |
Library strategy: RibosomeProfiling
Basecalls were performed using RTA version 2.4.11
For processed data files, NGSutils verson 0.5.9 was used to remove sequencing adapters and filter out any reads shorter than 25 bp. Fastx version 0.0.13 was subsequently used to remove the first base from each read and resulting reads were aligned to the NCIB 3610 genome (NZ_CP020102.1) using Bowtie version 1.1.2. Reads that uniquely aligned to the genome were assigned to a single position corresponding to the 15th nucleotide from the 3’ end.
Genome_build: Bacillus subtilis NCIB 3610 (NZ_CP020102.1)
Supplementary_files_format_and_content: Wig files with the first column containing the chromosome position and the second column containing the number of reads that mapped to that position.
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| Submission date |
Feb 07, 2019 |
| Last update date |
May 21, 2019 |
| Contact name |
Daniel B. Kearns |
| E-mail(s) |
dbkearns@indiana.edu
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| Organization name |
Indiana University
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| Department |
Biology
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| Lab |
Kearns Lab
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| Street address |
1001 E 3rd St
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| City |
Bloomington |
| State/province |
IN |
| ZIP/Postal code |
47405-7005 |
| Country |
USA |
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| Platform ID |
GPL24109 |
| Series (2) |
| GSE126234 |
Suppressor mutations in ribosomal proteins and FliY restore Bacillus subtilis swarming motility in the absence of EF-P [Ribosome Profiling] |
| GSE126235 |
Suppressor mutations in ribosomal proteins and FliY restore Bacillus subtilis swarming motility in the absence of EF-P. |
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| Relations |
| BioSample |
SAMN10884313 |
| SRA |
SRX5347634 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3594064_DK5955_B_fwd.wig.gz |
379.0 Kb |
(ftp)(http) |
WIG |
| GSM3594064_DK5955_B_rev.wig.gz |
367.0 Kb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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