eamag/protocols-with-steps huggingface dataset:
TITLE: Tissue preparation, immunohistochemistry, Imaging, and Quantification
AUTHORS: Bryan  Yoo, Jessica  Griffiths, Sarkis  Mazmanian

[DESCRIPTION]
Protocol for neuronal imaging used in Yoo et al 2021

[STEPS]
SECTION: Tissue Preparation
1. 100mg/kg of pentobarbital (Euthasol - Virbac, Carros, France) was administered intraperitoneally (IP), and tissues were perfused with 30mL of phosphate buffer solution (PBS) and next with cold 4% paraformaldehyde (PFA) in PBS. 

SECTION: Tissue Preparation
2. GI tract was post-fixed in 4% PFA overnight at 4°C, and stored in PBS + 0.025% sodium azide. 
SECTION: immunohistochemistry
3. Tissues that underwent subsequent immunohistochemistry were made transparent by use of the passive CLARITY technique (PACT) (Treweek et al., 2015). 
SECTION: immunohistochemistry
4. Briefly, perfused and fixed tissues were embedded with polymerized 4% (wt/vol) acrylamide, and lipids were eliminated using 8% (wt/vol) SDS solution.
SECTION: immunohistochemistry
5. Tissues were blocked in 3% donkey serum and permeabilized with PBS + 0.3% Triton (PBST). 
SECTION: immunohistochemistry
6. Primary antibodies were incubated in PBST for 48 hours and washed with PBST for 24 hours (replacing wash 3 times). Primary antibodies used were rabbit PGP9.5 (1:300; Millipore Sigma AB1761-I) and chicken GFAP (1:500, BioLegend Cat #829401). 
SECTION: immunohistochemistry
7. Tissues were next incubated in secondary antibodies (and DAPI) for 24 hours and washed in PBS for 48 hours, intermittently replacing the wash solution with fresh PBS. Secondary antibodies used were goat anti-chicken Alexa 647 (Life Technologies A-21450) and donkey anti-rabbit Alexa 568 (Life Technologies A10042). 
SECTION: Imaging
8. Tissues imaged just for virally expressed, endogenous fluorescence were made transparent using sorbitol-based optical clearing method, ScaleS (Hama et al., 2015).
SECTION: Imaging
9. Tissues were mounted in respective mounting mediums (RIMS and Scales S4), on a glass slide with a 0.5mm spacer (iSpacer, SunJin Lab Co.). 
SECTION: Imaging
10. Images were acquired on Zeiss LSM 780 or 880, and microscope, laser settings, contrast, and gamma remained constant across images that were directly compared. 
SECTION: Imaging
11. All confocal images were taken with the following objectives: Fluar 5× 0.25 M27 Plan-Apochromat 10× 0.45 M27 (working distance 2.0 mm) and Plan-Apochromat 25× 0.8 Imm Corr DIC M27 multiimmersion.
SECTION: Quantification
12.  Neurons in each ganglion were counted by counting cells that are of a distinct color. Large intestinal ganglia were defined as distinct if separated by a width of 3 or fewer neurons.
TITLE: sample_prep_urine.nan
AUTHORS: NAN KB, Mario Uchimiya, John Glushka, Leandro I Ponce, Saraa Al Jawad, Laura Morris, Arthur Edison

[DESCRIPTION]
This is a modified protocol for NMR metabolomics for urine samples. This method was originally proposed by:
 
See also:

 




[BEFORE_START]
This protocol assumes that the original urine samples have >600 µL.

[STEPS]
20. 
SECTION: Day-1/1
1.8. Add 600 µL of 100% cold methanol to 300 µL of samples on ice 
Use 1.5-mL tubes

SECTION: Day-1/1
1.7. Vortex the samples for 10 s

SECTION: Day-1/1
1.6. Incubate the samples at -20 °C for 20 min 

SECTION: Day-1/1
1.5. Centrifuge the samples at   for 30 min 

SECTION: Day-1/1
1.4. Transfer the supernatant of each sample to a new tube
Use 1.5-mL tubes
SECTION: Day-1/1
1.1. Dry the samples in a speed-vac concentrator  

 

SECTION: Day-1/1
1.9. Thaw samples on ice or at 4 °C 
1. Day-1/1
1.1. Thaw samples on ice or at 4 °C 
1.2. Centrifuge the samples at 4 °C at 12000 x g for 5 min

 


1.3. Add 60 µL of the phosphate buffer to 540 µL of the supernatant for each sample

Use 1.5-mL Eppendorf tubes
1.4. Vortex the samples at 4 °C for 2 min
1.6. Transfer 590 µL of the supernatant to an NMR tube for each sample
 


SECTION: 2
1. 
SECTION: 2
1.2. Dry the samples in a speed-vac concentrator  

SECTION: 2
1.3. 
1.5. Centrifuge the samples at 4 °C for 10 s
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<h2>Introduction</h2>
<p>The incidence of thyroid cancer is increasing and histological test by itself cannot differentiate thyroid cancer from some benign nodules. Our immediate goal is to meta-analysis and determines the impact of promoter methylation of eight selected candidate TSGs on thyroid cancer and to identify the most important molecules in this carcinogenesis pathway.</p>
<h2>Methods and analysis</h2>
<p> We will include observational studies evaluating the promoter methylation in patients with thyroid cancer. A comprehensive search was performed using PubMed, Scopus, and ISI Web of Knowledge databases, and eligible studies were included. The methodological quality of the included studies was evaluated according to the Newcastle Ottawa scale table and pooled odds ratios (ORs); 95% confidence intervals (CIs) were used to estimate the strength of the associations with Stata 12.0 software. Egger’s and Begg’s tests were applied to detect publication bias, in addition to the “metatrim” method.</p>
<p><strong>Ethics and dissemination</strong></p>
<p>No ethical issues are predicted. These findings will be published in a peer-reviewed journal and presented at national and international conferences.</p>
<p><strong>Registration number</strong></p>
<p>This systematic review protocol is registered in the PROSPERO International Prospective Register of Systematic Reviews, registration number (CRD42016033484).</p>
<p><strong>Strengths and limitations of this study</strong></p>
<p>This systematic review, for the first time, will conduct to evaluate the prognostic and diagnostic accuracy of DNA methylation in patients with thyroid cancer using comprehensive search of several databases. The study screening, data extraction, and risk of bias assessment of the current study will be conducted by two researchers independently. We expect some potential heterogeneities between previous studies, including stage, and histological grade in patient samples.</p>

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE:  FSCV and Ephys Recording Setup
AUTHORS: Raymond Murray, Helen Schwerdt

[DESCRIPTION]
Methods detailing the recording setup using both electrophysiological (Ephys) and fast-scan cyclic voltammetry (FSCV) are described. 

[STEPS]
SECTION: Recording Setup
1. One to four selected probes were utilized to record dopamine concentrations using fast scan cyclic voltammetry (FSCV) while electrophysiological (Ephys) activity was recorded from all other implanted probes not connected to FSCV. Probes used to record dopamine in a given recording session were switched to record local field potentials (LFPs) in later recordings sessions to allow for recording both types of signals from the same site, and vise versa for probes originally recording LFPs. 
SECTION: Recording Setup
2. A current-to-voltage converting head stage circuit was employed on probes recording FSCV, allowing the recording of current during concurrent application of a triangle wave via the FSCV system. 
SECTION: Recording Setup
3. Remaining probes were alternatively connected to a voltage-follower head stage circuit for Ephys recording to be amplified and digitized using the Digital Lynx SX system from Neuralynx.
SECTION: Recording Setup
4. Ag/AgCl electrodes surgically implanted in the animal's epidural tissue or brain matter were grounded by the FSCV system, also serving as the FSCV and Ephys reference. The Ephys system ground was connected to the experimental rig. 
SECTION: Recording Setup
5. FSCV scans consisted of applying a triangle wave to each probe at a rate of 400 V/s at a frequency of 10 Hz. Specifically, each scan lasted 8.5 ms consisting of an initial ramp from -0.4 V to + 1.3 V, then returning -0.4 V. Color plots were generated by background subtracting currents at a specific timepoint and plotting scan voltage on the y-axis, time on the x-axis, and current change amplitude as color. 
SECTION: Recording Setup
6. Dopamine redox was identified according to current changes at -0.2 and 0.6 V relative to the reference Ag/AgCl electrodes during each scan. 
SECTION: Recording Setup
7. Principal component regression analyses and correlation to dopamine and pH standards recorded from an in vitro flow cell environment were performed to ensure recorded currents were mostly related to dopamine redox. 
SECTION: Recording Setup
8. The Ephys system recorded voltage inputs with a ± 1 mV range at 30 kHz with an applied bandpass filter from 0.1 Hz to 7500 Hz. 
SECTION: Recording Setup
9. This system also recorded time stamps of identified task events utilizing parallel ports that conveyed 8-bit event codes. Software functions and a messaging system used concurrently with the Ephys system  allowed for common time stamp output between the Ephys and FSCV systems.
TITLE: Soil Bioassay
AUTHORS: Andreea S

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Neuropeptide-like proteins (NLPs) are a type of neuropeptides that influence and regulate the neuronal behaviour of the nematodes. Altering of the levels of the NLPs lead to a changed behavioural output. With this soil bioassay experiment, the behaviour of the </span><span style = "font-style:italic;">Globodera pallida</span><span> towards the root exudate of the potato plant is studied in the presence and absence of a NLP secreting biofilm. This chemotactic experiment is retrieved from the paper of Farnier and et.al. from 2012. </span></div></div>

[STEPS]
?. [Soil Bioassay]
Two medium sized,  tubs of top diameter approximately   are to be used for this experiment which will assess the responses of second stage infective juveniles responses to chemical stimuli. One is to contain a young potato plant cultivar which secretes the neuropeptide-like proteins (NLPs) from the biofilm surrounding the roots. The other, to contain a regular young potato plant cultivar without the NLP secreting biofilm as a control set up. The stimuli, Potato Root Exudate (PRE) is secreted by the root of the potato plant. This side of the tub is hereafter refered to as the stimulus side (S-side).
10 L
?. [Soil Bioassay]
Second stage juvenile nematodes were released in the centre of the tub in a  droplet of water, about   away from the S-side. The number of nematodes in this drop will not be determined.
10 µl
?. [Soil Bioassay]
After  , a section of the soil is to be lifted and removed from the tub with a razorblade and a spatula.
?. [Soil Bioassay]
Since the number of J2s inoculated into the tub will not be known, the proportion of J2s found in the different sections is to be calculated based on the total number of nematodes recovered.
?. [Soil Bioassay]
The experiment will be repeated for each batch of nematodes.
?. [Soil Bioassay]
Tubs will be kept in darkness at room temperature, and nematodes will be given  to choose their direction of movement.
?. [Soil Bioassay]
The J2s will be recovered by re-suspension and separation in   of water in   vials.
5 mL
12 mL
?. [Soil Bioassay]
The supernatant above each section of soil will be poured into a counting-chamber for determination of the number of J2s under a Leitz stereo microscope. Nematodes recovered from the S-sides of tub will be considered to have been attracted, nematodes in the central section will be considered to have expressed no-choice, and nematodes around the spot of their release will be considered not attracted to potato metabolites.
TITLE: Visium CytAssist FFPE v3 -- University of Minnesota TMCs
AUTHORS: Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
The Visium CytAssist Spatial Gene Expression for FFPE assay is designed to analyze mRNA in
tissue sections derived from formalin fixed & paraffin embedded (FFPE) tissue samples. The Visium
CytAssist instrument requires a glass slide with intact tissue sections as input. 

Tissue sections are processed with Visium Spatial Gene Expression assay performed by the University of Minnesota Imaging Center and sequenced by the University of Minnesota Genomics Center.



[STEPS]
SECTION: Deparaffinization, H&amp;E Staining, Imaging &amp; Decrosslinking
1.   

SECTION: Library Preparation &amp; Sequencing
2.   

 


SECTION: FASTQ Generation
3. BCL data from Illumina sequencer is demultiplexed and converted into FASTQ format using bcl2fastq software version 2.20.0
TITLE: Protocol 2: CRISPR Transformation
AUTHORS:  

[STEPS]
?. Mix Bacterial transformation solution with cultured E coli. Pipette 100uL of Bacterial Transformation mix into a microcentrifuge tube. Use an inoculation loop to lightly scrape E coli and transfer to the microcentrifuge tube. Use pipetting technique of aspirating and dispensing to homogenize the mixture.
?. Add CRISPR reagents. Retrieve tubes of Cas9 Plasmid, gRNA Plasmid, and Template DNA. Add 55uL of sterile water to each. Don’t forget to change the pipette tip. Make sure they are mixed well. In this order, pipette 10 uL from Cas9 Plasmid, gRNA Plasmid, and Template DNA into a centrifuge tube. Add each to the bottom of the competent cell mixture tube and follow by flicking your wrist or tapping the tube to the table to mix after each reagent addition.
?. Place the mixture into a PCR tube.
?. Incubate the tube in the refrigerator for 30 minutes.  While you are waiting for the prepare the following:
?. LB media. Add 1.5 mL of room temperature water to the 1.5mL microcentrifuge tubes containing LB Media. Add 500 uL first and mix by pipetting so the tube doesn't overflow. Once the mixture is completely dissolved, add 250 uL of the LB media into a new microcentrifuge tube.
?. On the bento lab, create a custom heating with PCR option. Make a setting where the PCR rack heats up to 42℃ for 30 seconds.
?. Take the tube out of the refrigerator and place in the PCR rack for 30 seconds.
?. Place contents of the PCR tube into the microcentrifuge tube containing 250 uL of LB Media.
?. Incubate for 6-12 hours.  If room temperature is cool, let the tube incubate for a longer period of time. If room temperature is warm, let the tube incubate for a shorter period of time.
TITLE: PCR and gel electrophoresis 
AUTHORS: Ashwinuday  

[DESCRIPTION]
This protocol can be used to confirm genes or DNA of interest from a template using PCR. Also to amplify required amount of genes to larger amounts.

[STEPS]
1. Prepare the working stock solution of all the reagents required. If already prepared and stored, takeout from the refrigerator and thaw on ice.

2. Prepare the following mixes each of total 50 µL : -  

 


3. Perform PCR using a Thermo cycler with the following temperature settings (All temperatures are in degree Celcius) :

 
Set the 2nd, 3rd and 4th steps to repeat 30 times (or maximum till 35)
 

4. 
 

To prepare the 1% agarose gel, mix 0.5 g of agarose in 50 mL of 1X TAE buffer and heat well to form a clear solution. 


 

5. Then the mix is allowed to cool till it is bearable to touch. Carefully add 2 µL of EtBr to the mix and swirl well.

6. Then into a set gel cast with the appropriate comb (which is decided by the number of wells and the size of the wells required), pour the mix without bubbles. 
 

7. Once the gel solidifies, carefully take it out with a gloved hand and place in the buffer tank with enough buffer to cover the immersed gel and remove the comb without breaking the wells.
8. Mix approximately 10 µL samples with 1X loading dye and load into each gel. Load a 1kb ladder (or any suitable ladder) into the first lane. Run the gel at 100V

9. After the dye front crosses 75%of the gel length, stop the current.
10. Take the gel out (with gloved hands) by letting all the buffer out into the tank and then image the gel on UV.
Compare the sample bands with the ladder to estimate the bands of interest
TITLE: 城市微生物相調查計畫—採集土壤教學
AUTHORS: Hsin-Mao  Wu

[DESCRIPTION]
吳昕懋

[STEPS]
SECTION: 如何加入專案
1. 開啟Line app，將頁面切換成「聊天」，並按下頂部的掃描圖示(紅框處)
  

SECTION: 如何加入專案
2. 掃描以下QR code或點選此連結微生物公民科學
  

SECTION: 如何加入專案
3. 加入成功後會顯示以下畫面
  

SECTION: 如何加入專案
4. 接著等待採集工具包寄送即可開始採集！
SECTION: 採土與iNaturalist紀錄教學
5. 移除表土，取距離土表5~7公分之處的土，裝入塑膠夾鏈袋中  
  

SECTION: 採土與iNaturalist紀錄教學
6. 將塑膠袋中的土均勻混合，倒入採集罐之中  
 


SECTION: 採土與iNaturalist紀錄教學
7. 在採集紀錄紙上勾選採樣地點、採樣種類，以及填寫日期、城市、鄉鎮、地點、採集者等資訊
SECTION: 採土與iNaturalist紀錄教學
8. 最後拍兩張照片
 

    
    

 

SECTION: 採土與iNaturalist紀錄教學
9. 接著打開iNaturalist app
SECTION: 採土與iNaturalist紀錄教學
10. 上傳完成後，在詮釋資料中找到ID(如下圖紅框處)  
 


SECTION: 採土與iNaturalist紀錄教學
11. 在採集紀錄紙上填入ID，並將紀錄紙下半部，沿虛線撕下並放入採集罐中   

SECTION: 採土與iNaturalist紀錄教學
13. 將採集罐保存於室溫，並於10天內以郵信寄回高雄醫學大學生物醫學暨環境生物學系
SECTION: 採土與iNaturalist紀錄教學
9.1. 如圖所示，點選右下角的加號  

SECTION: 採土與iNaturalist紀錄教學
9.2. 如下圖所示接著需選擇觀察類型，請點「選擇照片」   

SECTION: 採土與iNaturalist紀錄教學
9.3. 請點選左上角紅框處的相機圖示，上傳第二張照片；接著點選圖中央綠框處，定位採集地點  
 

SECTION: 採土與iNaturalist紀錄教學
9.4. 按下打勾處完成定位  

SECTION: 採土與iNaturalist紀錄教學
12. 記得將採集地點恢復原狀！
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Polyploidization contributes to the complexity of gene expression resulting in numerous related but different transcripts. This study explored the transcriptome diversity and complexity of tetraploid Arabica coffee (Coffea arabica) bean. Long-read sequencing (LRS) by Pacbio Isoform sequencing (Iso-seq) was used to obtain full-length transcripts without the difficulty and uncertainty of assembly required for reads from short read technologies. The tetraploid transcriptome was annotated and compared with data from the sub-genome progenitors. Caffeine and sucrose genes were targeted for case analysis._x000D_&lt;br&gt; An isoform-level tetraploid coffee bean reference transcriptome with 95,995 distinct transcripts (average 3,236 bp) was obtained. A total of 88,715 sequences (92.42%) were annotated with BLASTx against NCBI non-redundant plant proteins, including 34,719 high quality annotations. Further BLASTn to NCBI non-redundant nucleotide sequences, C. canephora coding sequences with UTR, C.arabica ESTs and Rfam resulted in 1,213 sequences without hits, were potential novel genes in coffee. Longer UTRs were captured, especially in the 5'UTRs, facilitating the identification of upstream ORFs (uORFs). The LRS also revealed more and longer transcript variants in key caffeine and sucrose metabolism genes from this polyploid genome. Long sequences (&gt;10kb) were poorly annotated._x000D_&lt;br&gt;</p>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol for western blot to detect strep-tagged constructs, using chromogenic detection with Strep-Tactin AP (Alkaline Phosphatase) conjugate. Based on protocol from iba life sciences.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
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?.
TITLE: simpleISM - A straight forward guide to upgrade from confocal to ISM
AUTHORS: Monalisa Goswami, René Lachmann, Robert Kretschmer, Rainer Heintzmann

[DESCRIPTION]
A hands-on guide to convert a confocal microscopy (here Olympus FluorView 300) into an image scanning microscopy (ISM) using a fast CMOS camera (here Andor Neo) as the detector. 

[STEPS]
1. 
Replace the dichromatic beam splitter slider of the confocal microscope (Supplementary Figure 1) with a custom-made slider that has a mirror at a 45-degree angle to reflect the beam out of the confocal microscope and a hole to let the beam pass and get detected with PMT channel 2. The slider will be used to switch between confocal and ISM.

3. Drill a hole in the microscope body to let the beam out of the microscope using the above-mentioned mirror slider (Supplementary Figure 2).


  

4. Using a mirror slide as a sample, align the reflected laser beam onto the camera (Andor Neo sCMOS camera is used here) by running the Fluoview software in ‘Point’ scan mode. Use a telescope to project the pinhole plane onto the camera. 
5. On the camera, select the smallest possible region of interest (ROI) (like 12x12) and align the focal spot to the centre of the ROI. 
5.1. Intercepting the clock pulses from Fluoview 300 Hardware
Optional Step: The Olympus Fluoview 300 laser scanning confocal microscope has two computers, the ‘power supply unit’ (PSU) and the ‘trigger unit’(TU) (Supplementary Figure 3). To observe the ramp-like scan signal, connect pin 11 and pin 15 of the DB-15HD connector (situated at the back of the TU) to the. On the Fluoview software, set the scan area to ‘512x512’ and zoom to ‘10’. Run the software in ‘XY repeat’ mode.



  

SECTION: Provide synchronization using Arduino Due
6. Prepare a subtractor circuit using the circuit diagram shown in Figure 3 in the paper. Use an Arduino Due board and connect it as shown in Supplementary Figure 4.


  

SECTION: Measuring ISM
7. Align the sample to the focus of the objective using the PMT detector channel 2. 
SECTION: Measuring ISM
8. Once the sample comes into focus, set the scan area to ‘512x512’ and zoom setting to ‘10’. Stop the scanning and toggle the switch to ISM and the slider at the mirror position.
SECTION: Measuring ISM
9. Upload the provided Arduino code. Run the Fluoview software in “XY repeat” mode and acquire the camera images using the settings shown in Table 1 in the paper.
SECTION: Measuring ISM
10. Once the camera acquisition is done, stop the “XY repeat” mode in the Fluoview software and save the images on the camera in the .fits format. 
SECTION: Measuring ISM
11. Acquire a background image following steps 8 and 9. 
SECTION: Measuring ISM
12. Use the ISM reconstruction code to get the final super-resolved ISM image.
2.
TITLE: Tache_Yuan_OT2OD024899_CLARITYAnd3DImagingOfColonicENSintheMouseAndPig_1_2019-Mouse_Protocol (Annotation Copy)
AUTHORS: Pu-Qing Yuan, Lixin Wang, Yvette Taché

[STEPS]
?. The male adult C57BL/6J mice (6-8 weeks, 25.4-28.6g, n=4) were used for CLARITY by PARS (perfusion-assisted agent release in situ) technique, a method for whole-body clearing and immunolabeling. The mice were perfused transcardially with 10 ml of ice cold PBS followed by 20 mL of the ice cold hydrogel solution at a slow rate of about 1 mL/min. Colonic samples (about 2-3 cm long) were removed from the proximal (0.5 cm from the cecum) and distal colon (1 cm from the anus). The colon was opened along the mesenteric border, pinned flat in Sylgard-coated dish and incubated in the ice cold hydrogel solution overnight for hydrogel-tissue hybridization. On the second day, the flat samples were cut into small pieces in 1.5x1.5 cm, keep the samples at 4°C for more 2 days in the hydrogel solution. Then the samples (6-8 pieces) were moved into 50 ml conical tubes with 15 mL of fresh hydrogel solution. Place the conical tubes on a rack in the desiccation chamber for degassing followed by a hydrogel polymerization in a temperature-controlled 37°C water bath. The sample were removed in a new 50 ml conical tube with 40 mL of clearing solution to wash out the excess hydrogel monomers from inside the tissue at 37°C overnight on a shaker/rotator plate until clearing was achieved in 1-2 weeks.  Immunofluorescence of PGP 9.5 (1:1000, ab108986, Abcam) and double labeling of neurofilament 200 (NF) with neuronal nitric oxide synthase (nNOS) (1:500, sc-648, Santa Cruz), S100β (1:500, ab52642, Abcam) with Hu C/D (1:200, A-21271, Life Technologies) were performed. Samples were incubated in primary antibody solution at room temperature (RT) with shaking for 2 days and washed with PBS at RT with shaking for 1 day followed by incubation in secondary antibodies at RT with shaking for 1 day. Samples were immerse samples in RIMS at RT for 3 hours and 4°C overnight and them mounted with RIMS in a sealed watertight well prepared with iSpacers (SunJin Lab, Hsinchu City, Taiwan).  Images acquired with ZEISS LSM710 confocal and SP8 DIVE multi photon microscope were reconstructed into 3D images and videos using Imaris 9.1 for neuroscientists.