eamag/protocols-with-steps huggingface dataset:
TITLE: Body Asymmetry
AUTHORS: Cristina Zogmaister, Federica Durante, Silvia Mari, Franca Crippa, Chiara Volpato

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes a measure of asymmetry of images of body shapes.</div><div class = "text-block">Two judges have separately measured the asymmetry of our set of photographs portraying men and women.</div><div class = "text-block">The inter-rater reliability for ABPI was excellent, r = .94 (α = .97). </div></div>

[STEPS]
TITLE: Colorimetric RT-LAMP Research group SARS-COV-2 detection protocol
AUTHORS: Teodor Emilov Mentov

[STEPS]
?. [vortex swab sample]
Vortex swab sample in Viral transport medium
?. [vortex swab sample]
Take 200µl and transfer to 1,5ml microtube
?. [vortex swab sample]
Add 500ul of ethanol reconstituted lysis  buffer incubate 10 minutes. Vortex or shake after
?. [vortex swab sample]
Add solubilization buffer, and wait 2 minutes, shake or vortex after
?. [vortex swab sample]
Add 200µl 98% Ethanol shake and use a an intense and brief centrifuge. 5-10 sec to 30 000g. This will avoid clogging in the filter cartrige afterwards.
?. [vortex swab sample]
Transfer the supernatant to the Kurabo catrige.
?. [vortex swab sample]
Wash with wash buffer and filter 3 times with Kurabo-Mini480.
?. [vortex swab sample]
add  50-100 (better 50)µl of elution buffer or pcr grade water.
?. [vortex swab sample]
filter and you have extracted the viral RNA in the elution tubes. You may store it in -80C or use it directly in the isothermic amplification
?. [vortex swab sample]
Isothermal amplification: Make a mix of 5µl of eluate sample and 5µl of pcr grade H2O and 2,5 µl of 10x primer mix in a 1,5ml transparent microtube.  Standart primer concentrations of 1·6 μM FIP/BIP, 0·2 μM F3/B3, 0·4 μM FL/BL work just fine.
?. [vortex swab sample]
add 12.5 µl of colorimetric RT-LAMP master mix. The NEB one or your own. Mix well with the pipette tip.
?. [vortex swab sample]
add 50µl of mineral oil to seal the reaction. This is essencial. Unsealed reactions are very difficult to detect.
?. [vortex swab sample]
Make a photo with your mobile phone. to register base colour, should be red-pink.
?. [vortex swab sample]
incubate 30 minutes at 63Cº. Take a photo afterwords. You dshold see the positive reactions as yellow. You can register it measuring the green channel in the RG colourspace (a normalized RGB colourspace). Higher than 83 is positive, loyer or equal is negative. To do this take the median values of the RGB measure with ImageJ, divide R,G,B to R+G+B. That is RG colorspace. You only  need the green channel.
TITLE: E. coli growth curve assay in BG-11 + sucrose
AUTHORS: sanjana  

[DESCRIPTION]
To obtain growth curves of KJK01 and pCSCX-KJK01 in BG-11 + sucrose and to characterize their growth. This also serves to estimate sucrose consumption and butanol production over time.

This can be adapted for any other bacteria as well by giveing proper growth conditions, and using appropriate wavelength to measure the OD and giving proper time intervals based on whether the bacteria is slow growing or fast growing.


[STEPS]
SECTION: Primary culture
1. Inoculate 1 colony of E. coli KJK01 in 5 mL  of 1X LB

SECTION: Primary culture
2. Inoculate 1 colony of E. coli pCSCX-KJK01 in 5 mL of 1X LB with 5 µL  Kan50 and 5 µL Amp100

SECTION: Primary culture
3. Leave both cultures in a shaker incubator   at 37 °C  and   

SECTION: Secondary culture
4. The next morning, inoculate 900 µL  of 1° culture of KJK01 in 90 mL  BG-11 and 10 mL of sucrose. This serves as a control for sucrose consumption.

 

SECTION: Secondary culture
5.  Inoculate 900 µL  of 1° culture of pCSCX-KJK01 in 90 mL  BG-11 and 10 mL of sucrose with 90 µL Kan50 and 90 µL Amp100. This serves as a control for butanol production.
 


SECTION: Secondary culture
6. Inoculate 900 µL  of 1° culture of pCSCX-KJK01 in 90 mL  BG-11 and 10 mL of sucrose with 90 µL Kan50, 90 µL Amp100 and 10 µL  IPTG (0.1 Milimolar (mM) )

 

SECTION: Secondary culture
7. Place these three cultures in a shaker incubator at 37 °C  and   
SECTION: Preparing samples
8. At 360 min intervals, draw 21 mL samples from each culture

SECTION: Preparing samples
9. Measure the OD of 1 sample from each culture at 600 nm and note it down for the growth curve assay  
SECTION: Preparing samples
10. Centrifuge all 6 samples at 5000 rpm, 7 °C for 15 min 

SECTION: Preparing samples
11. Decant the solution and store the supernatants at -20 °C 

SECTION: Preparing samples
11.1. Submit 1 sample of each culture for the sucrose assay. This helps determine consumption of sucrose over time.
SECTION: Preparing samples
11.2. Submit 1 sample of each culture for NMR analysis. This helps determine butanol production over time.
SECTION: Preparing samples
12. Continue this procedure for 5760 min
TITLE: LEGACY01: STUDY MANAGEMENT
AUTHORS: Katrina M Pollock, Calliope Dendrou

[DESCRIPTION]
This protocol details study management in an experimental medicine study of seasonal influenza vaccination responses in Lymph nodE single-cell Genomics in AnCestrY (LEGACY01).

[GUIDELINES]
STUDY MANAGEMENT

The day-to-day management of the study will be co-ordinated through NIHR Imperial CRF and the study manager Aime Palomeras.

LABORATORY DATA RECORDING AND MONITORING

Laboratory data management for the study will be co-ordinated by Calli Dendrou, Brian Marsden and Teresa Lambe at the University of Oxford. Data will be stored with linked anonymised (pseudonymised) codes, and all files and databases will be password protected and held on password protected computers. Pseudanonymised identifiers will be kept on a secured NHS workstation at NIHR Imperial CRF before transfer of the minimally required demographic metadata (e.g. sex and age) to study scientists in Oxford.

Raw and analysed sequencing, genetic, flow cytometric and serological data will be collated and stored on a restricted access site on a University of Oxford computational cluster (BMRC) and will be managed and curated according to established protocols. The storage system of the computational cluster is resilient to disk failure and data are automatically backed up to linked off-site servers. Data files will be clearly labelled and associated (cleaned and anonymised) metadata will be stored in tab-delimited text files so as to document the provenance of the data. Where automated analyses are performed, these will be version controlled and the commands used will be recorded in log files. Data arising from smaller-scale laboratory analyses will be recorded in hard copy or electronic laboratory notebooks according to standard University practices. Data (and analysis scripts) will be maintained in this way for ten years after study completion.

PROJECT MANAGEMENT AND GOVERNANCE

Imperial College London and University of Oxford are the principal partners for delivery of the study in the UK. These two institutions will partner with Uganda Virus Research Institute (UVRI), who will be responsible for international capacity building to undertake future single-cell and lymphatic tissue focussed vaccine research under the umbrella of the LEGACY Network. 

[STEPS]
TITLE: Single-cell ATAC sequencing
AUTHORS: Klaus H. Kaestner Lab, Suzanne Shapira

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Interrogating cell-type specific chromatin accessibility can reveal cis-regulatory elements linked to downstream gene expression patterns. Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq) is a technique that assays genome-wide chromatin accessibility and can reveal nucleosome positioning patterns, map enhancer and promoter regions, or reveal transcription factor binding sites. Single-cell Assay for Transposase-Accessible Chromatin using Sequencing (scATAC-seq) adapts the bulk ATAC-sequencing protocol to separate individual nuclei and assay chromatin state with single-cell resolution. This technique allows for resolution of gene accessibility patterns within specific pancreatic cell types such as α-cells, β-cells, and acinar cells.</span><span></span></div><br/><br/><br/></div>

[STEPS]
?. [Steps in pre-processing]
1. Transfer handpicked islets (approximately 5,000 IEQs) into  conical tube. 2. Add  of 1xPBS w/o Ca2+, Mg2+ (Rockland, MB-008). Centrifuge for 2 min at RT, 180 xg. Aspirate the supernatant.3. Add  of warm ( ) 0.05% Trypsin (Invitrogen, 25300054) to the islets. Pipette up and down with p1000. 4. Incubate at  for 9 min, or until cells are in single cells. Pipette up and down at t=7 min, 4 min, 2 min, 0 min. 5. Stop the trypsin reaction by adding  of 100% FBS (Hyclone, SH3091003) to the dissociated islets and pass cells through BD FACs tube with strainer top (Corning 352235)6. Use  of 100% FBS to rinse the tube and pass through the strainer.7. Transfer cells to  conical. Centrifuge 4 min, 400 xg. 8. Remove the supernatant and wash cells with PBS with 10% FBS. Centrifuge for 4 min, 400 xg. 9. Wash the cells with PBS with 10% FBS and centrifuge for 4 min, 400 xg. Remove the supernatant.10. Count cells using a countess chamber.11. For the scATACseq, do the final resuspension in 0.04% BSA in PBS as is consistent with the instructions for using fresh cell in the Nuclei Isolation Protocol.
15 mL
10 mL
1 mL
37 °C
37 °C
1 mL
1 mL
15 mL
?. [Links to kits used in post-processing]
1. For nuclei isolation the protocol used is: Nuclei Isolation for Single Cell ATAC Sequencing. We target a 5000 nuclei recovery for this protocol.2. Samples are processed for scATAC seq using Chromium Single Cell ATAC Reagent Kits.
TITLE: Perfusion/ Fixation for cryostat slicing for HHC
AUTHORS: louis-eric.trudeau

[DESCRIPTION]
This protocol details the Perfusion and Fixation for cryostat slicing for HHC.

[STEPS]
SECTION: Cryostat slicing
1. Weight the animal.

SECTION: Cryostat slicing
2. Anesthetize the animal with sodium pentobarbital (100 mg/kg  et  7 mg/ml) by i.p. injection with a 27G needle.

SECTION: Cryostat slicing
3. Confirm the absence of pain reflex (paw pinch).

SECTION: Cryostat slicing
4. Perfuse the animal with 50 mL of PBS followed by 50 mL of PFA 4% by trans-cardiac injection with a butterfly needle while the right atrium is detached from the superior vena cava.

SECTION: Cryostat slicing
5. Extract the brain and immerse it  2880 min  in PFA 4% followed by 2880 min-5760 min in sucrose 30% (until the initially floating brain drops to the bottom of the tube)

Composition:
 

SECTION: Cryostat slicing
6. Using dry ice, cool isopentane to -30 °C and submerge the brains for  15 s-30 s in the freezing solution before storing them at -80 °C.
TITLE: Transformation
AUTHORS: Shuning    Guo

[DESCRIPTION]
This protocol is used to transform plasmid DNA into competent cells by chemical method or electroporation.

[BEFORE_START]
Do not place the competent cells on the the ice for too long time before start.

[STEPS]
1. Choose one transformation method depending on your requirement of transformation efficiency.
2. Wash the 0.1 or 0.2 cm electroporation cuvette (store in 75% ethanol)by tap water for one time and ddH2O for two times and dry in the oven.
3. Place the electroporation cuvette on the ice and use UV to disinfect for 20 min.
4. Mix 25μl electrocompetent cells with 1 to 2 μl of DNA (total DNA concentration less than 10ng/mL, maximum 25ng). Mix well and incubate on ice for ~1 min.
5. Set the MicroPulser to "Ec1" when using the 0.1 cm cuvettes. Set it to "Ec2" or "Ec3" when using the 0.2 cm cuventtes.
6. Transfer the mixture of cells and DNA to a cold electroporation cuvette and tap the suspension to the bottom. Place the cuvette in the chamber slide. Push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber. Pulse once.
7. Remove the cuvette form the chamber and immediately add 1 ml of SOC medium to the cuvette. Quickly but gently resuspend the cells with a eppendoff.
 

8. Transfer the cell suspension to a 1.5 ml polyproylene microfuge tube and incubate at 37℃ for 1 hour, shaking at 200 rpm.  

9. Check and record the pulse parameters. The time constant should be close to 5 milliseconds.
10. Plate on selective LB or SOB medium and inverted culture for 14~16 hours.
2. Thaw the competent cells on the ice for 10 min to let the suspension thaw.
3. Mix 100μl competent cells with about 10μl DNA. 
4. Incubate on ice for ~30 min.
7. 
Add 0.5~1ml LB medium into the mixture of cells and DNA and mix well.
8. Incubate at 37℃ for 1 hour, shaking at 200 rpm.
9. Plate on selective LB medium.
5. Heat shock at 42℃ for 45 seconds in the water bath.
6. Incubate on ice for 2~5 min.
TITLE: SynGO analysis
AUTHORS: Chuyu Chen

[DESCRIPTION]
synapse function and gene enrichment analysis

[STEPS]
SECTION: SynGO analysis
1. Convert mouse Gene ID to human genes symbols with ID conversion tool 
SECTION: SynGO analysis
2. Copy converted genes symbols to the SynGO input section, select background as brain expressed, and name the experiment in the description 
SECTION: SynGO analysis
3. Start analysis and export data to Excel files
SECTION: SynGO analysis
4. Open custom color-coding of SynGO ontologies
SECTION: SynGO analysis
5. Provide GO term ID and -log10(GSEA 'gene cluster' FDR corrected p-value) in the input section, label for figure legend, and three values to create a color gradient suitable for the data. 

 
SECTION: SynGO analysis
7. Download figures
SECTION: SynGO analysis
6. Open a color-picker to change the color-coding of the data
TITLE: Yeast cells live fluorescence imaging
AUTHORS: Florian  Wilfling, Fabian  Fiedler, Anna  Bieber, Cristina    Capitanio

[DESCRIPTION]
Protocol for sample preparation and live fluorescence imaging of yeast cells. 

[STEPS]
SECTION: Yeast cell culture in low fluorescence medium
1. Grow yeast cells overnight in Low Fluorescence (LoFlo) medium.
SECTION: Yeast cell culture in low fluorescence medium
1.1. In the morning dilute cells in LoFlo to OD 0.1.
SECTION: Yeast cell culture in low fluorescence medium
1.2. Grow until mid-log phase (0.5–0.8 OD).
SECTION: Pre-treat microscope slides with Concanavalin A solution
2. Add 10 µl Concanavalin A to the glass surface.
SECTION: Pre-treat microscope slides with Concanavalin A solution
2.1. Let the slides dry completely before use.
SECTION: Prepare the sample for imaging
3. Once the cell culture has reached 0.5–0.8 OD, spin down 2 ml of the original culture. Resuspend the pellet in 30 µl LoFlo to obtain a concentrated sample. 
SECTION: Prepare the sample for imaging
3.1. Apply 3.5 µl of the concentrated yeast culture on the Concanavalin A-treated glass slide.
SECTION: Prepare the sample for imaging
3.2. Cover the sample with a glass coverslip.
SECTION: Imaging procedure:
4. First, choose a group of cells to use to set up adequate imaging settings. Use lasers corresponding to the right fluorophores and regulate exposure time based on the brightness of the features of interest. 
SECTION: Image analysis
5. Count cells and analyse signal with Fiji Cell counter Plug-in.
SECTION: Imaging procedure:
4.1. Set up stack acquisition. For yeast cells set a total stack size of 6-8 µm. We used a step size of 300 nm. 
SECTION: Imaging procedure:
4.2. In order to have an unbiased approach, use Transmitted Light to select a new a field of view for data acquisition. 
The field of view should contain 25-100 cells in the same focal plane. 
SECTION: Imaging procedure:
4.3. Acquire stacks.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
QIAgen's DNeasy Blood &amp; Tissue kit (cat#69504 or 69506) for cultured cells

[BEFORE_START]
<strong>Preparation of buffer AW1 and AW2</strong><br />Add ethanol to AW1 and AW2 as directed on bottle. Stable for one year.<br /><br />If buffer AL has precipitates, warm to 56C until precipitates dissolved<br /><br />Preheat thermoixer to 56C<br /><br /><strong>Equipment required:</strong><br />Pipettes and pipette tips<br />Vortexer<br />Microcentrifuge tubes, 1.5-1.7mL<br />Thermomixer for heating at 56C

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Immunofluorescent Staining of Foxp3 in Frozen Sections
AUTHORS: Sam Li

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">T regulatory cells (also known as Tregs or Regulatory T cells) are essential cells in the immune system that suppress immune responses of other cells, designed to limit excessive reactions and prevent autoimmunity. Tregs are characterized by the expression of CD4, CD25, and FOXP3, while lacking CD127. CD4+FOXP3+ regulatory T cells have been referred to as "naturally-occurring" regulatory T cells to distinguish them from "suppressor" T cell populations that are generated in vitro. While other variants of suppressive T cells do exist, such as CD8 suppressor cells, Th3 and Tr-1 cells, Tregs are classically defined as CD4+CD25+FOXP3+ cells. Here, we provide a protocol for immunofluorescent staining of FOXP3 in frozen sections, adapted from a protocol from Dr. Matthias Hardtke-Wolenski, Medizinische Hochschule Hannover. This protocol has been successfully used on mouse spleen, liver, and skin sections.</div></div>

[STEPS]
?. Thaw frozen sections in a sealed environment to avoid damage by frozen water crystals (Approximately 1 hour).
?. Fix for 5 minutes in acetone (-20°C). Note: Do not use more often than 5 times.
?. Dry for 10 minutes at room temperature.
?. Incubate sections in TBS-T (50 mM Tris, 150 mM NaCl, adjust pH with HCl to 7.6 with 0.05% Tween 20) for 15 minutes.
?. Pretreatment: Perform antigen retrieval or enzyme digestion if needed.
?. Wash twice for 2 minutes each with TBS-T.
?. Optional: staining of surface marker can be performed before "Cleaning" step.
?. Cleaning: Incubate sections with 1% Triton X-100 diluted in TBS for 30 minutes at room temperature. This step will help to reduce background staining (longer incubation may be more effective, especially for sections thicker than 10 µm).
?. Normal Serum Blocking: Without washing, incubate sections directly with 5% normal mouse/rat/rabbit serum blocking solution for 30 minutes at room temperature.Note: Normal serum should be the same species of which the secondary antibody is raised.
?. Wash three times for 2 minutes each with TBS-T.
?. Endogenous Peroxidase Blocking: Incubate sections with 3% H2O2 in TBS for 10 minutes to block endogenous peroxidise (Recommended for liver sections, but optional for other tissues).
?. Wash 3 times for 2 minutes with TBS-T.
?. Primary Antibody: Incubate sections with primary antibody at its optimal dilution (Mouse FOXP3 Alexa Fluor® 647, clone MF-14, cat # 126408) in "antibody dilution buffer" for 30-60 minutes at room temperature.Note: Using a purified unconjugated antibody along with a secondary reagent may improve staining due to signal amplification.
?. Wash twice for 2 minutes with TBS-T.
?. Counterstain twice for 3 minutes each with DAPI.
?. Wash twice for 2 minutes with TBS-T.
?. Coverslip with Mowiol or mounting medium.
TITLE: One-pot native barcoding of amplicons v4 (LoCost)
AUTHORS: Josh Quick, Lauren Lansdowne

[DESCRIPTION]
This one-pot native barcoding protocol was developed in conjunction with Oxford Nanopore Technologies, New England Biolabs and BCCDC.

[STEPS]
1. In a new PCR strip-tube/plate set up the following reaction for each sample:


 

 


2. Incubate at room temperature for  15 min 
Incubate at 65 °C for 15 min 
Incubate on ice for 1 min 

3. In a new PCR strip-tube/plate set up the following reaction for each sample:

 

 


4. Incubate at room temperature for 20 min 
Incubate at 65 °C for 10 min
Incubate on ice for 1 min 
 

5. In a new 1.5 mLEppendorf tube pool all one-pot barcoding reactions together. 
 




6. Add 0.4x volume of SPRI beads to the sample tube and mix gently by either flicking or pipetting. For example add 96 µL SPRI beads to 240 µL pooled one-pot barcoding reactions.

 

7. Mix by vortexing and pulse centrifuge to collect all liquid at the bottom of the tube. Incubate for 5 min at room temperature.
8. Place on magnetic rack and incubate for 2 min or until the beads have pelleted and the supernatant is completely clear. Carefully remove and discard the supernatant, being careful not to touch the bead pellet.

9. Add 250 µL SFB and resuspend beads completely by pipette mixing. Pulse centrifuge to collect all liquid at the bottom of the tube and place on the magnet. Remove supernatant and discard.
 



10. Repeat steps 11.9 to perform a second SFB wash. Pulse centrifuge and remove any residual SFB.
 

11. Add 200 µL of room-temperature 70 % volumeethanol to bathe the pellet. Carefully remove and discard ethanol, being careful not to touch the bead pellet.
 



12. Pulse centrifuge to collect all liquid at the bottom of the tube and carefully remove as much residual ethanol as possible using a P10 pipette.
13. With the tube lid open incubate for 1 min or until the pellet loses it's shine (if the pellet dries completely it will crack and become difficult to resuspend).

14. Resuspend pellet in 30 µL 10 millimolar (mM) Tris pH 8.0, mix gently by either flicking or pipetting and incubate for 2 min.

15. Place on magnet and transfer sample to a clean 1.5 mL Eppendorf tube ensuring no beads are transferred into this tube.
TITLE: Protocol for Standard RNA Synthesis with Hi-T7 RNA Polymerase (NEB #M0658)
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. [Assemble the reaction]
Thaw the necessary kit components, mix and pulse-spin in microfuge to collect solutions to the bottom of the tubes.Assemble the reaction at  in the following order.
0 Room temperature
The total reaction volume is  .
?. [Assemble the reaction]
Add Nuclease-free H2O: X µl
?. [Assemble the reaction]
Add 10X Hi-T7 RNA Polymerase Reaction Buffer:
2 µl
Hi-T7 RNA Polymerase Supplied with 10X Hi-T7 RNA Polymerase Reaction Buffer formulated for optimal performance.
?. [Assemble the reaction]
Add NTPs: X µl
[each]
?. [Assemble the reaction]
Add Template DNA: X µl from  to
?. [Assemble the reaction]
Add RNase Inhibitor:
0.5 µl
[final]
?. [Assemble the reaction]
Add Hi-T7 RNA Polymerase (50 U/µl):
2 µl
?. [Incubation]
Incubate at  for .
50 °C
TITLE: Nanovesicles extraction
AUTHORS: Veerle Baekelandt

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Nanovesicles extraction (Exosomal isolation, SHSY-5Y)</span></div></div>

[STEPS]
?. seed cells for  in 3 x 15 cm dishes at a density of 20 x 106 cells per plate
?. wash cells of all the plates with 2 times  of 1x PBS
10 mL
?. Add  DMEM with 1% exosome depleted FBS to each plate for Preparation of exosome depleted FBS:Commercial FBS was filtrated through 0.22 µm filter and
10 mL
Centrifuge: 140000 34
?. collect medium. purify nanovesicles with differential centrifugation (see substeps)
?. (remove cells)
Centrifuge: 300 34, 15 min
?. decant supernatant and collect nanovesicles in   cell lysis buffer (for western blotting) or 50 µl PBS+ PI (for intact cell cross-linking)
50 µl
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?. (remove cell debris)
Centrifuge: 15000 34, 30 min
?. filter through 0.22 µm filter (filtration of apoptotic bodies)
?. Centrifuge: 140000 34, 180 min, 4 10