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Merge pull request #235 from NFDI4Microbiota/improve-spellchecker
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Update spellcheck.yml
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pmjklemm authored Sep 11, 2024
2 parents 639831d + ee19d81 commit 51cb471
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475,228 changes: 475,228 additions & 0 deletions .github/workflows/spellcheck.acronyms.whitelist

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102 changes: 102 additions & 0 deletions .github/workflows/spellcheck.whitelist
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@@ -0,0 +1,102 @@
NFDI
NFDIs
Microbiota
mb
html
css
RDM
DMPs
parasitology
README
Findable
Interoperable
SOPs
RNALater
Ruocco
HCl
cardinality
microbiome
curation
Parasitology
DMP
DMPs
amplicon
metagenomic
rhizosphere
Pseudomonas
aeruginosa
HISAT
Illumina
NovaSeq
TruSeq
ontologies
mRNA
bioinformatics
protistology
Omics
TDR
TDRs
funder
funders
reusability
RDMO
de
forschungsdaten
DFG
DFGs
zbmed
CoreTrustSeal
OpenAIRE
Microbiota's
Microbiotas
anonymization
england
Bioinformatics
DeCaF
METASTAVA
GRP
DSW
Programme
Curation
DCC
DMPonline
Cessda
maDMPs
RDMkit
DSW
RDC
Actionability
AOS
Aruna
GDWG
NBI
scalable
IOM
FSD
al
et
engelhardt
pauls
pavone
rehwald
subdiscipline
Jupyter
reproducibility
Sielemann
sielemann
bres
FAIRsharing
Orthologous
macromolecular
omics
Bielefeld
ENVO
ENVOs
DDBJ
ENA
KnowledgeBase
NCBI
ELN
IRB
IRB
PAO
4 changes: 3 additions & 1 deletion .github/workflows/spellcheck.yml
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Expand Up @@ -15,4 +15,6 @@ jobs:
# The checkout step
- uses: actions/checkout@v3
- uses: rojopolis/[email protected]
name: Spellcheck
name: Spellcheck
with:
config_path: .github/workflows/spellcheck_config.yml
6 changes: 5 additions & 1 deletion .spellcheck.yml → .github/workflows/spellcheck_config.yml
Original file line number Diff line number Diff line change
Expand Up @@ -4,6 +4,9 @@ matrix:
lang: en
d: en_US
dictionary:
wordlists:
- .github/workflows/spellcheck.whitelist
- .github/workflows/spellcheck.acronyms.whitelist
encoding: utf-8
pipeline:
- pyspelling.filters.markdown:
Expand All @@ -12,6 +15,7 @@ matrix:
ignores:
- code
- pre
- a
sources:
- '**/*.md'
default_encoding: utf-8
default_encoding: utf-8
2 changes: 1 addition & 1 deletion README.md
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Expand Up @@ -8,7 +8,7 @@ This knowledge base is a collection of documents and references important to the

## Contributing to the Knowledge Base

If you want to contribtute to the Knowledge Base, please see the [02-contributing.md](https://github.com/NFDI4Microbiota/nfdi4microbiota-knowledge-base/blob/main/docs/_Getting-Started/02-contributing.md) file, and be sure to add yourself to the [03-contributors.md](https://github.com/NFDI4Microbiota/nfdi4microbiota-knowledge-base/blob/main/docs/_Getting-Started/03-contributors.md) file.
If you want to contribute to the Knowledge Base, please see the [02-contributing.md](https://github.com/NFDI4Microbiota/nfdi4microbiota-knowledge-base/blob/main/docs/_Getting-Started/02-contributing.md) file, and be sure to add yourself to the [03-contributors.md](https://github.com/NFDI4Microbiota/nfdi4microbiota-knowledge-base/blob/main/docs/_Getting-Started/03-contributors.md) file.

## License

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Expand Up @@ -26,47 +26,47 @@ CAA (alkylating buffer)
400 mM 2-chloroacetamide (CAA) in 50 mM HEPES/NaOH pH 8.5 or Milli Q (add 500 µl)

Cell Lysis and Protein Preparation
1. Add 100 µl of ice cold NP40-Lysis buffer to a cell pellet of 1*106 HEK293T cells and resuspend the cell pellet by pipetting up and down.
2. Sample A: transfer 36 µl to 1.5 ml Eppendorf tube and add 1.5 µl of benzonase. Incubate sample on ice until Sample B is prepared.
3. Sample B: Equilibrate a 0.45 µm filter plate with 50 µl plate wash buffer. Place filter plate on 96well plate and spin down for 2 min at 1.200 rpm at 4 °C. Place filter plate on new 96well plate and transfer the remaining NP40 lysate to the equilibrated filter plate. Centrifuge (500 g, 10 min, 4°C). Transfer 36 µl of the filtrate to a 1.5 ml Eppendorf tube. Add 1.5 µl benzonase and incubate sample for 5 min on ice.
4. Add 2 µl of 20% SDS to Sample A and B and incubate both lysates for 10 min at 37°C while shaking.
1. Add 100 µl of ice-cold NP40-Lysis buffer to a cell pellet of 1*106 HEK293T cells and resuspend the cell pellet by pipetting up and down.
2. Sample A: transfer 36 µl to 1.5 ml Eppendorf tube and add 1.5 µl of benzonase. Incubate the sample on ice until Sample B is prepared.
3. Sample B: Equilibrate a 0.45 µm filter plate with 50 µl plate wash buffer. Place the filter plate on a 96well plate and spin down for 2 min at 1.200 rpm at 4 °C. Place the filter plate on a new 96well plate and transfer the remaining NP40 lysate to the equilibrated filter plate. Centrifuge (500 g, 10 min, 4°C). Transfer 36 µl of the filtrate to a 1.5 ml Eppendorf tube. Add 1.5 µl benzonase and incubate sample for 5 min on ice.
4. Add 2 µl of 20% SDS to samples A and B and incubate both lysates for 10 min at 37°C while shaking.
5. Add 2 µL of 200 mM DTT (10 mM final concentration) and incubate for 20 minutes at 45°C.
6. Add 2 µL of 400 mM CAA (20 mM final concentration) and incubate for 20 minutes at 24°C.
7. Add 1 µL of 200 mM DTT to quench the reaction.

SP3 Protein Clean-up
Always prepare 70% ethanol fresh and get also acetonitrile fresh from the stock bottle.
1. Transfer 10 µl of lysate (~10 µg of protein) to a PCR tube.
2. Add 40 µl of 50 mM HEPES to the lysate, followed by 2 µl of beads, mix them well by pipetting.
2. Add 40 µl of 50 mM HEPES to the lysate, followed by 2 µl of beads, and mix them well by pipetting.
3. Immediately add acetonitrile to obtain a final percentage of 50%, (52 µl).
4. Incubate for 8 minutes at room temperature off the magnetic rack.
5. Place on magnetic rack and incubate for further 2 minutes at room temperature.
4. Incubate for 8 minutes at room temperature of the magnetic rack.
5. Place on a magnetic rack and incubate for further 2 minutes at room temperature.
6. Remove and discard supernatant, on the magnet.
7. Add 200 µL of 70% ethanol and incubate for 15 seconds on magnetic stand. Remove and discard supernatant.
8. Add 200 µL of 70% ethanol and incubate for 15 seconds on magnetic stand. Remove and discard supernatant.
9. Add 180 µL of acetonitrile and incubate for 15 seconds on magnetic stand. Remove and discard supernatant (all droplets) and air dry beads for 30-60 seconds.
7. Add 200 µL of 70% ethanol and incubate for 15 seconds on a magnetic stand. Remove and discard supernatant.
8. Add 200 µL of 70% ethanol and incubate for 15 seconds on a magnetic stand. Remove and discard supernatant.
9. Add 180 µL of acetonitrile and incubate for 15 seconds on a magnetic stand. Remove and discard supernatant (all droplets) and air dry beads for 30-60 seconds.
10. For digestion, reconstitute beads in 10 µL of digestion solution (e.g. 50mM HEPES pH 8.0 + 200 ng of trypsin (1:50 enzyme to substrate ratio). It is not necessary to completely homogenize the beads, as they will be stuck to each other and excessive handling will generate bubbles. The cluster will degrade as proteins are digested.
11. Incubate for 14 hours at 37°C.
Digested peptides can be recovered from the beads by placing the tube on a magnetic rack and removing the supernatant containing them.

SP3 Peptide Recovery and Preparation for TMT
1. Vortex and spin your samples then sonicate the tubes 5 minutes in the ultrasound bath. Resuspend
beads after digest by vortexing and spin shortly.
2. Place tube on magnetic rack and incubate for further 2 minutes at room temperature.
3. Transfer supernatant into new PCR tube
4. Wash the tube with the beads with 10 µl 50 mM HEPES, spin down and place the tube again for 2 minutes on magnet. Note: do not use any amine containing buffer (e.g. TRIS) in this step, as amines will also react with the NHS-group of TMT-labelling reagent.
1. Vortex and spin your samples then sonicate the tubes for 5 minutes in the ultrasound bath. Resuspend
beads after digestion by vortexing and spinning shortly.
2. Place the tube on a magnetic rack and incubate for further 2 minutes at room temperature.
3. Transfer the supernatant into a new PCR tube
4. Wash the tube with the beads with 10 µl 50 mM HEPES, spin down, and place the tube again for 2 minutes on the magnet. Note: do not use any amine-containing buffer (e.g. TRIS) in this step, as amines will also react with the NHS group of TMT-labelling reagent.
5. Combine the supernatants in the new tube.
Recovered samples can be directly used for TMT labeling.

SP3 TMT-Labelling
1. Reconstitute the aliquot of TMT labelling reagent in 5 µL of acetonitrile and pipette mix.
1. Reconstitute the aliquot of TMT labeling reagent in 5 µL of acetonitrile and pipette mix.
2. Add 4 µl of one TMT label to the digestion mixture and pipette mix. Incubate for 30 min to 1 hour at room temperature.
3. Add 4 µl 5% hydroxylamine, pipette mix and incubate for 15 minutes at room temperature.
The isobaric tag serves as a ‘barcode’ that indicates from which sample a peptides derived. Therefore, you can now mix TMT labelled peptides in a 1:1 ratio or you can perform label checks on each sample separately to determine the labelling efficiency and also to compare the overall amount of the samples for later mixing.
3. Add 4 µl 5% hydroxylamine, pipette mix, and incubate for 15 minutes at room temperature.
The isobaric tag serves as a ‘barcode’ that indicates from which sample peptides derived. Therefore, you can now mix TMT labeled peptides in a 1:1 ratio or you can perform label checks on each sample separately to determine the labeling efficiency and also to compare the overall amount of the samples for later mixing.
4. Dilute sample with 40 µl Buffer A from OASIS workflow.
5. Pool all samples for TMT16 multiplexing into a new Eppendorf tube.
6. Wash PCR tube once more with 40 µl Buffer A and combine to the Eppendorf tube.
You need to reduce the acetonitrile concentration in the sample before starting the desalting, else the sample would not be able to bind to the C18 material and you lose it.
6. Wash PCR tube once more with 40 µl Buffer A and combine it with the Eppendorf tube.
You need to reduce the acetonitrile concentration in the sample before starting the desalting, else the sample will not be able to bind to the C18 material and you lose it.
Desalting the samples by OASIS

Buffer A
Expand All @@ -75,7 +75,7 @@ Buffer A
Buffer B
80% acetonitrile (CAN), 0.05% FA in water

Reverse phase clean-up step of peptide mix using OASIS HLB µElution Plate, which consist of a Hydrophilic-Lipophilic Balanced reverse phase sorbent. A vacuum is applied for each step:
Reverse phase clean-up step of peptide mix using OASIS HLB µElution Plate, which consists of a Hydrophilic-Lipophilic Balanced reverse phase sorbent. A vacuum is applied for each step:

Equilibrate the well of an OASIS plate:

Expand All @@ -97,7 +97,7 @@ Desalting step:
Sample elution & collection:
8. Place glass vial to collect eluate below OASIS well
9. Elute with 50 µl B
10.Elute with 50 µl B
10. Elute with 50 µl B
Dry samples in Speedvac and reconstitute peptides in 1% formic acid supplemented with 4% acetonitrile. Samples are now ready for the injection on a mass spectrometer

## References
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