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| Status |
Public on Aug 02, 2024 |
| Title |
output2B NNK |
| Sample type |
SRA |
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| Source name |
yeast
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
cell type: yeast
genotype: [psi-pin-] (MATa ade1-14 his3 leu2-3,112 lys2 trp1 ura3-52)
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| Treatment protocol |
Cells were thawed from −80 °C in 50 ml plasmid selection medium at OD = 0.05 and grown until exponential for 15 h. At this stage, cells were harvested and resuspended in 400 ml protein induction medium (-URA, 2% glucose, 100 μM Cu2SO4) at OD = 0.1. After 24 h the input pellets were collected, and cells were plated on -ADE-URA selection medium in 145-cm2 plates (Nunc, Thermo Scientific). Plates were incubated at 30 °C for 7 days. Finally, colonies were scraped off the plates with PBS 1x and harvested by centrifugation to collect the output pellets. Both input and output pellets were stored at −20 °C before DNA extraction. For each random library experiment, one input samples and three technical replicates of the output pellet were processed for sequencing.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Input and output pellets were thawed and resuspended in 1.5 ml extraction buffer (2% Triton-X, 1% SDS, 100 mM NaCl, 10 mM Tris pH 8, 1 mM EDTA pH 8), and underwent two cycles of freezing and thawing in an ethanol-dry ice bath (10 min) and at 62˚C (10 min). Samples were then vortexed together with 1.5 ml of phenol:chloroform:isoamyl 25:24:1 and 1.5 g of glass beads (Sigma). The aqueous phase was recovered by centrifugation and mixed again with 1.5 ml phenol:chloroform:isoamyl 25:24:1. DNA precipitation was performed by adding 1:10 V of 3M NaOAc and 2.2 V of 100% cold ethanol to the aqueous phase and incubating the samples at -20°C for 1 hr. After a centrifugation step, pellets were dried overnight at RT. Pellets were resuspended in 900ul resuspension buffer (10 mM Tris pH 8, 1 mM EDTA pH 8) and treated with 7.5 ml RNase A (Thermo Scientific) for 30 min at 37˚C. The DNA was finally purified using 30ul of silica beads (QIAEX II Gel Extraction Kit, Qiagen), washed and eluted in 30 ul of elution buffer. Plasmid concentrations were measured by quantitative PCR with SYBR green (Merck) and primers annealing to the origin of replication site of the PCUP1-Sup35N plasmid at 58 °C for 40 cycles.
The library for high-throughput sequencing was prepared in a two-step PCR (Q5 high-fidelity DNA polymerase, NEB). In PCR1, 300 million of molecules were amplified for 15 cycles at 68ºC with frame-shifted primers with homology to Illumina sequencing primers (Supplementary Data Primers). The products were purified with ExoSAP treatment (Affymetrix) and by column purification (MinElute PCR Purification Kit, Qiagen). They were then amplified for 12 cycles in PCR2 with Illumina-indexed primers.
Libraries of random sequences were synthesized by Integrated DNA Technologies (IDT) as ultramers of 12 NNK codons (36 nucleotides). Sequences were flanked by constant regions of 25 nt upstream and 21 nt downstream for cloning. The NNK ultramers were extended in a 1-cycle PCR (Q5 high-fidelity DNA polymerase, NEB). The resulting products were treated with 2ul/tube of ExoSAP (ExoSAP-IT, Applied Biosystems) for 30 minutes at 37 °C and 20 minutes at 80 °C and purified through a MinElute column (Qiagen). In parallel, the PCUP1-Sup35N plasmid was linearized by PCR (Q5 high-fidelity DNA polymerase, NEB). The products were purified from a 1% agarose gel (QIAquick Gel Extraction Kit, Qiagen) and ligated by Gibson with 3 h of incubation at 50°C (Supplementary Data Table 3 Gibson) followed by dialysis for 3 h on a membrane filter (MF-Millipore 0.025 μm membrane, Merck) and vacuum concentration. The resulting libraries were transformed into 10-beta Electrocompetent E. coli (NEB), by electroporation with 2.0 kV, 200 Ω, 25 μF (BioRad GenePulser machine). Cells were recovered in SOC medium for 30 min and grown overnight in 50 ml of LB ampicillin medium. A small amount of cells was also plated in LB ampicillin plates to assess transformation efficiency. Total transformants were estimated, 50 ml of overnight culture were harvested to purify each library with a midi prep (Plasmid MIDI Kit, Qiagen).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Description |
from plasmid library
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| Data processing |
FastQ files from paired-end sequencing of each library library before (‘input’) and after selection (‘output’) were processed using the DiMSum pipeline (https://github.com/lehner-lab/DiMSum)
Assembly: no genome used for assembling - Random Library
Supplementary files format and content: excel book (.xlsx). Processed sequencing data. Each sequence has nucleator status (1 o 0) and a mean nucleation score
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| Submission date |
Jun 25, 2024 |
| Last update date |
Aug 02, 2024 |
| Contact name |
Mariano Martín |
| E-mail(s) |
mmartin@ibecbarcelona.eu
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| Organization name |
IBEC
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| Street address |
c/ Baldiri Reixac 10-12
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| City |
Barcelona |
| ZIP/Postal code |
08028 |
| Country |
Spain |
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| Platform ID |
GPL31112 |
| Series (1) |
| GSE270792 |
Amyloids “at the border”: deep mutagenesis and random sequence extension reveal an incomplete amyloid-forming motif in Bri2 that turns amyloidogenic upon C-terminal extension |
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| Relations |
| BioSample |
SAMN42045122 |
| SRA |
SRX25057802 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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