eamag/protocols-with-steps huggingface dataset:
TITLE: Data collection
AUTHORS: Alise Ponsero

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocols aims to details the steps necessary to collect new data points for the "Database Justice League" project.</div></div>

[STEPS]
?. [Download data]
Once you have received the link to download your dataset, download the csv file locally.Make sure to save it on your computer in a convenient folder!In this protocol we will perform the data collection on a test dataset:
?. [Download data]
As you can see in this test dataset, some preliminary information have been collected automatically such as the article name, authors and publication date.However some information will need additional efforts to be collected, such as the database name, link and availability.
?. [Database name and link]
In the dataset, you'll find a column name "DOI", this is a usique identifier given to all scientific publications. This allows to easily find the article online.In order to access the paper search in you web browser :            https://doi.org/ + DOI numberIn the first example it would be: https://doi.org/10.1093/nar/gkaa818
?. [Database name and link]
The first information you need to collect is the name of the database! It seem trivial, but people use acronyms and are often not consistent in naming databases. So be careful!There is mainly two possibilities: either the article is presenting a new database or it is an article about an update of an already published database
?. [Database URL]
Next, we need to find the database URL. In general, the URL is mentionned in the article abstract.As an example for the first article in the test dataset, you see in the article abstract:"The NDB aims to be a shared resource to biologists, biophysicists and all genome scientists. The NDB is available at https://ndb.rice.edu."Here you need to report the full URL (https://ndb.rice.edu) under the "current_access" column.
?. [Database accessibility]
Finally, we need to assess if the URL for the database is working. Click on the URL you identified!Two possibilities: The URL is working or not!
?. [Database URL]
Next, we need to find if the database is accessible online, and if yes, what is its URL.Usually, the link will be displayed in the abtract (often at the end of the abstract) of the article. As an example in the first article of the test set:"The NDB is available at https://ndb.rice.edu."Report the URL (https://ndb.rice.edu) in the table under the "current_access" column
?. [Database accessibility]
Finally we need to assess if the database is accessible or not.To do so, first, click on the database URL that you have identified.
?. [Submit]
Great job! Your table should now look like this: You can upload the completed dataset to D2L.
?. [Submit dataset]
Great job! You collected all the data we needed to complete the DB Justice League! You should now have a table filled as follow:Upload the table on D2L and notify us of the upload!Thank you for your great contribution!
TITLE: Combinatorial selective ER-phagy remodels the ER during neurogenesis
AUTHORS: Melissa Hoyer, Harper JW, Ian R. Smith, Julia C. Paoli, Yizhi Jiang, Joao A. Paulo

[DESCRIPTION]
The endoplasmic reticulum (ER) has a vast proteomic landscape to perform many diverse functions including protein and lipid synthesis, calcium ion flux, and inter-organelle communication. The ER proteome is remodeled in part through membrane-embedded receptors linking ER to degradative autophagy machinery (selective ER-phagy). A refined tubular ER network is formed in neurons within highly polarized dendrites and axons. Autophagy-deficient neurons in vivo display axonal ER accumulation within synaptic ER boutons, and the ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy. However, mechanisms, including receptor selectivity, that define ER remodeling by autophagy in neurons are limited. Here, we combine a genetically tractable induced neuron (iNeuron) system for monitoring extensive ER remodeling during differentiation with proteomic and computational tools to create a quantitative landscape of ER proteome remodeling via selective autophagy. Through analysis of single and combinatorial ER-phagy receptor mutants, we delineate the extent to which each receptor contributes to both magnitude and selectivity of ER clearance via autophagy for individual ER protein cargos. We define specific subsets of ER curvature-shaping proteins or lumenal proteins as preferred clients for distinct receptors. Using spatial sensors and flux reporters, we demonstrate receptor-specific autophagic capture of ER in axons, which correlates with aberrant ER accumulation in axons of ER-phagy receptor or autophagy-deficient neurons. This molecular inventory of ER proteome remodeling and versatile genetic toolkit provides a quantitative framework for understanding contributions of individual ER-phagy receptors for reshaping ER during cell state transitions.

[STEPS]
TITLE: Come importare identificatori OAI-PMH in OpenRefine
AUTHORS: Andrea Marchitelli

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocollo per importare in OpenRefine una lista di identificatori OAI-PMH per successive elaborazioni.</div></div>

[STEPS]
?. Aprire OpenRefine e scegliere "Crea progetto"
?. Selezionare "Indirizzo/i Web (URLs)" come fonte dei dati del nuovo progetto e cliccare su "Avanti". Inserire la baseurl OAI-PMH del target individuato, scegliere il verbo ListIdentifiers e il metadataPrefix opportuno.Es. http://www.bibliotecheoggi.it/trends/oai?verb=ListIdentifiers&metadataPrefix=oai_dc
Il protocollo OAI-PMH consente l'impostazione di limitatori per data e per set per esporre dataset parziali e permettere raccolte incrementali.Es. http://an.oa.org/OAI-script?verb=ListIdentifiers&from=1998-01-15&metadataPrefix=oldArXiv&set=physics:hep
?. Selezionare "Files XML" nella sezione "Analizza i dati come"
?. Nell'area dell'anteprima del file da importare, individuare la prima riga contenente l'elemento  e portare il puntatore del mouse a evidenziarla.Poi cliccarvi sopra.
?. A quel punto si sarà ottenuta la lista degli identificatori OAI-PMH, uno per riga, pronta per elaborazioni successive.
TITLE: projectpr1d_metab.nan
AUTHORS: NAN KB, John Glushka, Mario Uchimiya, Christopher Esselman, Saraa Al Jawad, Leandro I Ponce, Laura Morris, Arthur Edison

[DESCRIPTION]
This is a protocol for running the Bruker pulse program "projectpr1d".

This pulse program was originally proposed in:

 


[BEFORE_START]
This protocol assumes your sample is loaded, locked, tuned, and shimmed in the magnet.

[GUIDELINES]
This protocol intends to provide concise instructions to carry out the experiment. For more comprehensive information, see Bruker's documentation including "Basic NMR Experiments" by clicking ? →  Manuals (docs) on the menu bar on TopSpin.

[STEPS]
SECTION: Create a new dataset
1. 
SECTION: Create a new dataset
1.1. On the menu bar on TopSpin, click on
Start → Create Dataset
 
 

SECTION: Create noesypr1d experiment file
0.29. When Lock, Tune, Shim are complete proceed to the specific protocol for your desired pulseprogram(s).
SECTION: Create a new dataset
1.4. A new window opens. On the right top bar, select
Source = /opt/NAN_METAB/par

In the list, select the one you want to use:
PROJECTPR1D_br600_serum.par: Parameter set optimized for serum and plasma samples.

 


Click
OK
 

SECTION: Create a new dataset
1.2. Enter
NAME: Name of a set of datasets (e.g., human_serum_study1). Use a single string.
EXPNO: Dataset number. Use a positive integer.

Select
Directory: Your directory.
 


 

SECTION: Create a new dataset
1.3. Select
Read parameterset

Click the button
Select
29. 
29. 
28.1. pulsecal
27.2. rga
26.3. zg
28.1. 
SECTION: Acquire a spectrum
0.29. 
SECTION: Acquire a spectrum
2. Go to the "USE DEFAULT" tab below to proceed with the default optimized parameters.

3. 
3. 
3.1. Load the calibrated P1 using the following command in the command line.
 
(e.g., getprosol 1H 10.01 -7.45)

 

3.1. Select the AcquPars tab to show a parameter set.

 
 

3.4. Common additional parameters to adjust:
 

3.2. Click on
Acquire → Gain
in the menu bar to automatically set the receiver gain.

 
 

3.3. Click
Go
in the menu bar to acquire a spectrum.

 

3.4. Click on
Process → Proc. Spectrum
in the menu bar to execute an automated processing macro.

 
3.2. Click the icon
_∏ _
to see a complete parameter set.

 

3.5. Acquire a spectrum. If more optimization is needed,  .
SECTION: Create a new dataset
1.5. Click
OK
3.5. If you want to modify parameters to improve your spectrum,   and move to a step case "MODIFY PAR".

3.3. Change parameters as needed.

Typically, the main parameter to adjust is NS:
TITLE: Stern REHAB
AUTHORS: Abby Myers

[DESCRIPTION]
Instructions for taking care of Stern's REHAB vials.

[STEPS]
SECTION: Adding stocks to Stern REHAB
1. When a vial looks sick, first check all other copies (including drobot copy) and if EVERY vial looks sick, consolidate all of the flies in a fresh food vial with moist cotton roll
1.1. If not all vials look sick, top off your sick vial with the other healthy copy

2. Add vial with consolidated flies and all older vials to designated box (organized by species) on REHAB cart next to Stern HF carts
SECTION: Flipping REHAB (every Friday)
3. Flip flies from vial in front to a fresh vial with a moist cotton roll

SECTION: Adding stocks back after REHAB
7. 
If vial is healthy and ready to go back, add one vial to its spot in the HF boxes
(These are organized by species and in ascending order of stock ID)
SECTION: Adding stocks back after REHAB
8. Add other vial back to Drobot the next time its species is being flipped (Drobot 1)
SECTION: Adding stocks back after REHAB
7.1. These are organized by species and in ascending order by stock ID
1.2. Cotton rolls are located on the back of the REHAB cart underneath the purple REHAB vials
SECTION: Flipping REHAB (every Friday)
4. Check vials behind the first one. If flies have emerged, flip those into the new vial as well
SECTION: Flipping REHAB (every Friday)
5. If vial is looking healthy, separate into two vials and wait another week. If both are healthy by the end of the next week, ready to add back to Drobot/HF box.
SECTION: Flipping REHAB (every Friday)
6. Discard oldest vial if it looks like nothing else will emerge
TITLE: Ligate 3′ RNA Adapter (On-Bead)
AUTHORS: Eric L. Van Nostrand, Thai B. Nguyen, Chelsea Gelboin-Burkhart, Ruth Wang, Steven M. Blue, Gabriel A. Pratt, Ashley L. Louie, Gene W. Yeo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Profiling of RNA binding protein targets in vivo provides critical insights into the mechanistic roles they play in regulating RNA processing. The enhanced crosslinking and immunoprecipitation (eCLIP) methodology provides a framework for robust, reproducible identification of transcriptome-wide protein-RNA interactions, with dramatically improved efficiency over previous methods. Here we provide a step-by-step description of the eCLIP method, along with insights into optimal performance of critical steps in the protocol. In particular, we describe improvements to the adaptor strategy that enables single-end enhanced CLIP (seCLIP), which removes the requirement for paired-end sequencing of eCLIP libraries. Further, we describe the observation of contaminating RNA present in standard nitrocellulose membrane suppliers, and present options with significantly reduced contamination for sensitive applications. These notes further refine the eCLIP methodology, simplifying robust RNA binding protein studies for all users.</div></div>

[STEPS]
?. [Prepare RNA Adapter Ligation Master Mix]
Prepare ;  per sample:  AB1H2O9 μL210× Ligase buffer (no DTT)3 μL30.1 M ATP0.3 μL4100% DMSO0.8 μL550% PEG 80009 μL6Murine RNase Inhibitor0.4 μL7High concentration T4 RNA Ligase2.5 μL
on ice
25 µl
AB1H2O9 μL210× Ligase buffer (no DTT)3 μL30.1 M ATP0.3 μL4100% DMSO0.8 μL550% PEG 80009 μL6Murine RNase Inhibitor0.4 μL7High concentration T4 RNA Ligase2.5 μL
?. [Prepare RNA Adapter Ligation Master Mix]
Mix carefully by pipetting (do not vortex) (see Note 3).
?. [Perform RNA Adapter Ligation]
Add  to each sample.
[RNA adapter ligation master mix]
?. [Perform RNA Adapter Ligation]
Add  to each sample.
[InvRiL19 RNA adapter]
?. [Perform RNA Adapter Ligation]
Incubate at  for ; flick to mix every ~
0 Room temperature
TITLE: TS Spurrs - cell pellet (TM - 013)
AUTHORS: sandra.crameri

[DESCRIPTION]
This method is used for conventional processing of cell pellets to Spurr's resin.

[GUIDELINES]
All time are minimum times, it is acceptable to go over time specified for any given step. Good place steps to leave overnight or at 70% ethanol and 50/50 Spurrs/Ethanol mix.

[STEPS]
SECTION: HEADER
1. SAN:


SPEC No:


OPERATOR & STEPS:


OPERATOR & STEPS:
SECTION: CONVENTIONAL
2. 2.5 % volume   for at least 40 min 

SECTION: CONVENTIONAL
3. Wash 0.1 Molarity (M) Sorenson's Phosphate Buffer pH 07.2 (300mosmol/kg) for 15 min 

SECTION: CONVENTIONAL
4. 1 % volume   in buffer for 60 min 

SECTION: CONVENTIONAL
5. 70 % volume   for at least 20 min 

SECTION: CONVENTIONAL
6. 95 % volume   for 20 min 

SECTION: CONVENTIONAL
7. 100 % volume   for 20 min 

SECTION: CONVENTIONAL
8. 100 % volume   dried molecular sieved for 20 min 

SECTION: CONVENTIONAL
9. 50 % volume   50 % volume   for 30 min 


SECTION: CONVENTIONAL
10. 100 % volume   30 min 

SECTION: CONVENTIONAL
11. 100 % volume   60 min 

SECTION: CONVENTIONAL
12. Oven polymerise overnight at 65 °C
TITLE: LUHMES culturing and differentiation protocol
AUTHORS: Mallory Wright, William J Buchser, ckremitz, jwaligor, bachman@wustl.edu

[STEPS]
SECTION: LUHMES coating protocol
1.  
0.1 mg/mL
50 µg/µL

SECTION: LUHMES Passaging (~ every 2-3 days)
20. Triturate cells only 1 or 2 times before seeding.
SECTION: LUHMES Passaging (~ every 2-3 days)
19. Discard the supernatant and resuspend in 1mL and use a 5mL pipette
SECTION: LUHMES Passaging (~ every 2-3 days)
18. Transfer cells to 15mL tube and centrifuge for 7min at 1200 RPM
SECTION: LUHMES Passaging (~ every 2-3 days)
17. Neutralize trypsin with 6mL of pre-warmed DMEM/F12
SECTION: LUHMES Passaging (~ every 2-3 days)
16. Add 4mL of the pre-warmed .025% Trypsin/EDTA   
and place in the incubator for 3 minutes.

SECTION: LUHMES Passaging (~ every 2-3 days)
15. Remove media and rinse with DPBS
SECTION: LUHMES growth media
14.  
       
 Add recombinant human FGF to media last
SECTION: LUHMES coating protocol
7.  
 Size: 100uL, Storage Temperature:  –20 °C in aliquots
1 mg/mL
2 µg/µL

SECTION: LUHMES growth media
13. Change (pre-warmed) media every 1-2 days
 

SECTION: LUHMES coating protocol
8. Slowly thaw the fibronectin solution at 2–8 °C. 
 

SECTION: LUHMES coating protocol
9. Dilute the Fibronectin in sterile Hank’s Balanced Salt Solution (HBSS). 
Add 2 µL to 998 µL 

SECTION: LUHMES coating protocol
11. Rinse 3 times with HBSS.
SECTION: LUHMES coating protocol
12. Air dry for 15 minutes and add LUHMES growth media. 
SECTION: LUHMES coating protocol
2. Thaw an aliquot of Poly-L-Ornithine solution at room temperature.
SECTION: LUHMES coating protocol
3. Dilute Poly-L-Ornithine solution to 50ug/mL in sterile water.
(Add 500 µL of PLO for every 500 µLsterile water)

SECTION: LUHMES coating protocol
4. Add 7mL of the 50ug/mL PLO to a T-75 overnight at RT.
SECTION: LUHMES coating protocol
5. Rinse 3 times with bio-grade water.
SECTION: LUHMES coating protocol
6. Allow the flask to air dry for 15 minutes uncapped and stand upright in the hood. (turn on UV)
SECTION: Cryopreservation
22. Label 2mL cryovials with the date, name, FIV#, Qbench number, passage number and cell type and add about 1.5 million cells per vial with 1mL freezing media. 

Freezing Media: 
 7mL LUHMES Media
 4uL B-fgf (40ng)
 2mL FBS (20%)
1mL DMSO (1%)
SECTION: LUHMES Passaging (~ every 2-3 days)
21. Add fresh culture media to flask for at least 15 min before seeding cells to
allow media to reach normal pH
SECTION: LUHMES coating protocol
10. Place fibronectin coated flask in incubator for 3 hours
SECTION: LUHMES Differentiation
23. Day 0, when the LUHMES are 80% confluent, add differentiation media and incubate overnight.
SECTION: LUHMES Differentiation
24. Day 1, replate cells onto a new poly-l-ornithine and fibronectin-coated plate and replace cells at a density of 1 million cells per T-75 flask or ~ 400,000 per well of a 6-well.
SECTION: LUHMES Differentiation
25. Replace LUHMES differentiation media with fresh differentiation media every day (neurons become mature in 7 days)
TITLE: LIVE IMAGING OF i3NEURONS (Support Protocol 5) 
AUTHORS: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Live imaging permits visualization of molecular and organellar dynamics within the neuron. While a standard confocal microscope is sufficient for short imaging experiments, extended imaging applications (>1 hr) are best served by a 37 °C live imaging chamber outfitted onto the microscope. CM should also be changed to Hibernate A Low Fluorescence Medium (BrainBits LLC, </span><a href="http://www.brainbitsllc.com/hibernate-a-low-fluorescence/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">cat. no. SKU#HAPR</span></a><span>) for extended imaging. </span></div></div>

[STEPS]
TITLE: Nutil Data Integration
AUTHORS: Michael X. Henderson

[DESCRIPTION]
This protocol describes Nutil data integration from segmentation and registration of mouse brain data.

[STEPS]
SECTION: Nutil Data Quantification
1. Open Nutil and find operation tab.
  
SECTION: Nutil Data Quantification
2. Find the 3 folders you created at the beginning of this workflow and populated during the workflow and populate them into their corresponding list above. (segmentation is your input folder, brain atlas map folder is your atlas folder, output folder is output and should be empty still XML or JSON anchoring file is the QuickN JSON file that you created earlier – you can also use visualign json file it doesn’t matter the anchor is set in quickNII and nonlinear adjustments are made in reference to it. Object color Is black because that’s what the staining of our segmentations look like if it were a different color we would need to look for another object color.
 
SECTION: Nutil Data Quantification
3. Navigate to advanced settings In the row labeled Global Pixel Scale/Unit, set it to 36.44778384 um2. (6.0 is new value for van andel scans) The NEW images that are scanned here at Van Andel are resolved at 0.5031 microns per pixel, so if we downsized by 12 we get 6.0372 then this number is squared to account for um2. the final value is 36.44778384.
SECTION: Nutil Data Quantification
4. Also, under advance settings change custom masking to yes and select the color of the hemisphere of interest i.e. white to view hemisphere masked in white mask. Also select the folder location of the mask. Repeat the color selection with black to view the other hemisphere masked by black.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A hallmark of legume plants is their ability to establish mutualistic symbioses with <em>Rhizobium</em> bacteria and arbuscular mycorrhizal fungi (<em>Rhizophagus irregularis</em>). These symbionts help in providing nutritional benefits to the host plants. Herein, we provide a protocol to inoculate the common bean for the induction of symbiont colonization. This protocol has been routinely used in our laboratory with high reproducibility.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Shipping wastewater samples to FDA-CFSAN
AUTHORS: Maria   Balkey

[DESCRIPTION]
This SOP provides guidance for shipping wastewater samples to the US FDA Center for Food Safety and Applied Nutrition (CFSAN), covering specific steps for registering samples with CFSAN and for the preparation of shipments (documentation, packaging, notifications). We recommend that samples are shipped in two batches, one batch after 3 months of sampling and one batch at the conclusion of your sampling (~6 months).

[BEFORE_START]
This protocol has three sections:


Section 1:  Submission of metadata 
Section 2:  Preparation of Shipping supplies
Section 3:  Sample shipment


Associated protocols:
https://www.protocols.io/view/ncbi-submission-protocol-for-sars-cov-2-wastewater-ewov14w27vr2/v5 for populating sample metadata. 


[STEPS]
SECTION: Submission of metadata
1. Fill out the BioSample custom wastewater template (extension of NCBI's Generic SARS-CoV-2: wastewater surveillance, v1.0 ) according to the NCBI submission protocol for SARS-Cov-2 protocol, once completed, send it to covidtrakr@fda.hhs.gov for registration.  Successful registrations will receive a notification confirming the sample registration, it will will include a list of CFSAN accessions.   

SECTION: Preparation of shipping supplies
2. Contact your Biosafety Officer at your institution and request instructions on how to ship wastewater samples. 
SECTION: Preparation of shipping supplies
3. Make sure adequate shipping materials are available for the preparation of your package. Prepare your shipment according to your current SOPs, check your institution and courier guidelines for the shipping of dangerous goods (wastewater samples with SARS-Cov-2) before attempting any shipment.
SECTION: Preparation of shipping supplies
4. Prepare the following documents for your package along your permits, government and/or institution requirements.
Spreadsheet with the list of samples and the description of shipment contents.
Any notes or comments regarding your package. 
SECTION: Preparation of shipping supplies
5. Label your samples accurately (tubes and caps).  Each sample (200 mL minimum) must be labeled with a CFSAN accession provided after completion of the metadata registration.  If necessary, use printed labels and secure them with transparent tape. 
SECTION: Preparation of shipping supplies
6. Wastewater samples shall be shipped in dry ice.
SECTION: Preparation of shipping supplies
7. The outer packing must be labeled accordingly. The full name and address of the sender and recipient must be included.
SECTION: Sample shipment
8. Once all the above requirements are completed, ship by express/next-day air.  Ship only on Tuesday, Wednesday or Thursday.  Ship the isolates to the address included below.


U.S. FDA CFSAN
Attn: Christopher Grim/Maria Hoffmann
5001 Campus Drive
RM 4E009
College Park, MD 20740
Christopher.Grim@fda.hhs.gov
Maria.Hoffmann@fda.hhs.gov
Phone:240-402-1080 (Maria) or 240-402-3582 (Chris)


SECTION: Sample shipment
9. Email tracking number to covidTrakr@fda.hhs.gov. Include in the notification the number of samples shipped and the corresponding list of sample identifiers.
TITLE: total RNA抽提
AUTHORS: 张 雪

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">在收集到生物材料之后，最好能即刻进行RNA制备工作。若需暂时储存，则应以液氮将生物材料急速冷冻后，储存于-80℃冷冻柜。在制备RNA时，将储存于冷冻柜的材料取出，立即以加入液氮研磨的方式打破细胞，不可以先行解冻，以避免RNase的作用。</div></div>

[STEPS]
?. 取材：收集10~40个所需时期的斑马鱼胚胎
?. 用1×PBS漂洗，5min×2
on ice
全程冰上操作，RNase-free枪头
?. 每管加入1ml TRIZOL试剂，放置冰上反复吹打，4°C，12000 rcf，离心10min
on ice
用力吹打的同时注意不要让液体溅出
?. 上清转移至新的EP管，冰上放置5min
on ice
?. 加入200µl 氯仿后，剧烈震荡15s，冰上静置3min
on ice
?. 4°C，12000rcf，离心15min
?. 将上层无色水相转移至新EP管中，加入等体积（约500µl）的异丙醇（RNA专用），混匀，-20°C静置20min
-20 °C
?. 4°C，12000g rcf，离心10min，去上清，留沉淀
?. 用1ml 75％乙醇（RNA专用无水乙醇加RNase-free H2O, 70%-80%均可）漂洗沉淀，4°C，7500g离心5min
Centrifuge: 7500 1324, 5 min, 4 10
?. 重复上一步骤，然后去上清，留沉淀
Centrifuge: 7500 1324, 5 min, 4 10
?. 室温倒置于纸上晾干，加20µl RNase-free H2O溶解
0 Room temperature
?. 1µl测浓度，其余-80°C贮存
-80 °C
?. 打开离心机，保持在4°C，12000 rcf
Centrifuge: 12000 1324, 30 min, 4 10