eamag/protocols-with-steps huggingface dataset:
TITLE: Multi-dimensional potential factors influencing COVID-19 vaccine booster acceptance and hesitancy among university academic community in Bangladesh: a cross-sectional comparative study
AUTHORS: dn.roy

[DESCRIPTION]
Despite the potential therapeutic benefits of primer vaccine dosage regimens, public perceptions of COVID-19 vaccine booster dose (VBD) acceptance and hesitancy vary among various sub-group populations. This study method designed to investigate COVID-19 vaccine booster dose acceptance and compares the multi-dimensional potential factors influencing VBD acceptance and hesitancy among university teachers and the student community in Bangladesh. This cross-sectional comparative study used a self-administered, anonymous, and validated multi-item questionnaire to rationalize the study’s outlined objectives. The questionnaire was deployed online using an online survey tool (Google forms) and conveniently sent to teachers and students in different public and private universities between 15thJune, 2022 and 15thAugust, 2022 using electronic collection methods (social media platform and emails) and following the STROBE guideline.A non-parametric data analytical tool called binary logistic regression was employed to explore the pattern of association between explanatory variables and the response variable. All the key assumptions related to binary regression analysis were examined to adjust the model suitability. Assumptions of binary logistic analysis were tested. Raw data were inserted into Microsoft Excel version 10 and imported to Statistical Package for the Social Science (SPSS) software. IBM-SPSS version 25 (RRID: SCR_016479)was used for analyzing the data. In this study analysis, p<0.05 was considered statistically significant.

[STEPS]
1.
TITLE: A step by step guide  to using Visual Field Analysis
AUTHORS: Mathilde Josserand, Bastien Lemaire

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In this protocol, we provide a step by step guide to using Visual Field Analysis (VFA) successfully. VFA is a python program based on DeepLabCut toolbox (Nath et al., 2019). Using our program, it is possible to score reliably the eye use, activity, and time spent in different zones of different animal species and experimental paradigms for more reproducible research.</div></div>

[STEPS]
TITLE: nf-100GMX-variant-summarizer
AUTHORS: Israel Aguilar Ordoñez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Nextflow pipeline used to count variants for the 100GMX project</div><div class = "text-block"> 'nf-100GMX-variant-summarizer' is a pipeline tool that counts variants in a VEPextended annotated VCF file.</div><div class = "text-block">This pipeline generates 3 outputs:</div><div class = "text-block">1) a TSV file with the total number of SNV and indels</div><div class = "text-block">2) a TSV file with per sample counts for variants of type SNV, indel, novel, worldwide singletons, clinvar, gwascat and pharmgkb</div><div class = "text-block">3) a PDF file with the number of discernible variants in sample groups of interest.</div><div class = "text-block"><span>Important note: input file must be previously annotated by </span><a href="https://github.com/Iaguilaror/nf-VEPextended" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://github.com/Iaguilaror/nf-VEPextended</span></a></div></div>

[STEPS]
?. [Branch A]
Project Counts
a) Count samples and raw stats for all samples.b) Give the total counts data.
?. [Branch B]
No filter countsDependencies:
a) Filter variants that are not in ClinVar, GWAS Catalog or PGKB.b) Give the total counts data.
?. [Branch B]
Novel countsDependencies:
a) Filter variants without a rsID.b) Give the total counts data.
?. [Branch B]
Worldwide singletons countsDependencies:
a) Filter variants that are singletons.b) Keep variants that have not frequencies in another population.
?. [Branch B]
ClinVar countsDependencies:
a) Filter variants harbored in CinVar.b) Give the total counts data.
?. [Branch B]
GWASCatalog countsDependencies:
a) Filter variants harbored in GWAS Catalog.b) Give the total counts data.
?. [Branch B]
PGKB countsDependencies:
a) Filter variants harbored in Pharm GKB.b) Give the total counts data.
?. [Branch B]
Merge tablesMerge tables of counted data.Dependencies:
?. [Branc C]
Define groupsDependencies:NONE
a) Count samples per group.b) Join in a file samples per group.
?. [Branc C]
Select world rareDependencies:
a) Filter variants according AF in gnomAD populations and NatMex.
?. [Branc C]
Extract discernible VCFDependencies:
a) Extract variants in other samples.b) Extract variants in local samples.c) Extract exclusive variants.
?. [Branc C]
Count and PlotDependencies:
a) Count the number of discernible variants per group and type in a TSV file.b) Plot number of discernible variants per group and type.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol provides an efficient and reliable technique for obtaining HMW DNA(&gt;1Mb) from animal blood. It accompanies:</p>
<p> </p>
<p>Huang Zhihai, Xu Jiang, Xiao Shuiming, Liao Baosheng, Gao Yuan, Zhai Chaochao, Qiu Xiaohui, Xu Wen, Chen Shilin</p>
<p> (2016): Supporting data for 'Comparative optical genome analysis of two Pangolin species Manis pentadactyla and Manis javanica'. GigaScience Database.</p>

[STEPS]
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TITLE: A simple and quick protocol for isolating human IgM antibodies from plasma using magnetic beads
AUTHORS: Sudhir Bhatia, Gudrun Baersch

[DESCRIPTION]
Magnetic beads are used in various applications, e.g. cell isolation, nucleic acid analysis and endotoxin elimination. Numerous groups are actively involved in the development of immunoglobulins for various research purposes. For example, many research projects require human IgM from human plasma. IgM are antibodies that appear in the first phase of infections with pathogens; their presence therefore indicates that the infection has only just begun. In addition, IgMs are often needed in research to determine their properties, and some groups are working to determine the properties of such antibodies for future therapeutic use. In all these cases, users need to isolate them from plasma. 
We have developed a protocol to isolate such IgM antibodies using magnetic beads. You can also use this protocol to isolate sterile antibodies.

[BEFORE_START]
Please read manual.
Prepare the chemicals to be used.

[GUIDELINES]
Ethical guidelines may apply, please check before starting experiments.

[STEPS]
3. Performing of the isolation:
The user should use 30µl magnetic beads for the isolation of IgM from 2000µl plasma (or corresponding ratios). The user should use the plasma in a 1:4 dilution (add 1500µl buffer solution to 500µl plasma (Tube B).

4. Leave the mixture at room temperature for 30 minutes in dark. Shake occasionally.
5. Place the tube in the Genekam magnetic rack for 2 minutes. During this step, the magnetic beads are attracted towards the magnet.
6. Carefully remove the liquid with a pipette tip without distrubing the magnetic pellet and collect it in a new tube alternatively one can tilt the magnetic rack to drop the fluid in waste collecting container while keeping the magnetic pellet in the tube. (This is only possible, if user is using Genekam magnetic rack as the tubes fits very tightly in this rack.)
7. Wash twice with buffer solution: 
To do this, add 1000µl of buffer solution (Tube B) to the beads, place the tube in the magnetic rack and pipette off the liquid after 2 minutes while the magnetic beads remain in the tube.
8. Elution with elution buffer:
To do this, add 100µl of elution buffer (Tube C) to the beads and incubate for 10 minutes. Place the tube in the magnetic rack and collect the liquid in a fresh sample tube after 2 minutes. Finally, add 25µl of stop solution (Tube D).
9. The isolation of the IgM antibodies should be confirmed using ELISA. 
2. The user must first wash the specified quantity of magnetic beads (Tube A) in 1000µl buffer solution (Tube B). For this purpose, 1000µl buffer solution is added to the beads, the tube is placed in the magnetic rack and the liquid is pipetted off after 2 minutes while the magnetic beads remain in the tube. Resuspend the beads in corresponding amount of buffer solution (30µl Tube B). 
1. 1. Label the tubes and calculate the chemicals required.
TITLE: Ai14 Genotyping 
AUTHORS: Ronald.zegarra

[DESCRIPTION]
genotyping 

[STEPS]
1. Thaw reagent on ice for10 min

2. Prepare : 

 

3. Pipette 19 µL of   into 0.2 mL Tube 


4. Lightly Vortex DNA sample for 30 s

5. Add 1 µL of DNA to each tube 

6. Select "Ai14" PCR protocol on the thermocycler 
7. Run 15 uL of PCR samples and a DNA ladder on a 
 
 at 120 V for 60 min  or until the dye is somewhat in the middle of the gel.
TITLE: 04 Transformation
AUTHORS: TJUSLS China

[STEPS]
?. Preparation of competent cells1.Streak out the E.coli strain on an LBM plate (no ampicillin!) to isolate colonies and incubate at 37 degrees C overnight    (16-20 hours).2. Use a sterile inoculating loop to collect cells from a single colony and inoculate 50 ml sterile 1X LBM Grow at 37 degrees C overnight (16-20 hours) in a shaker incubator.3. Add 25 ml of the overnight culture to each 250 ml LBM flask.4. Grow the cultures to OD600 = 0.55.Decant supernatant and resuspend the cells in 1/4 original volume (87.5 ml) ice cold 100 mM MgCl2. Hold on ice for 5       minutes. Transfer the cells to pre-chilled sterile large centrifuge bottles. Spin in the for 10 minutes using the rotor 4000 rpm at 4 degrees C.6.Decant the supernatant and resuspend the cells in 1/20 original volume (17.5 ml) of ice cold 100 mM CaCl2. Hold on      ice for 20 minutes. Pellet as above 4000 rpm for 10 minutes.7.Decant the supernatant and resuspend the cell pellet in 1/100 original volume (3.5 ml) of a solution that is 85% v/v 100 mM CaCl2 and 15% v/v glycerol (100%). For each culture processed chill approximately 15 labeled eppendorf tubes in a dry ice-EtOH bath. Pipet 300 ul cells into each tube and place immediately into the dry ice-EtOH bath. 8.Transfer the frozen competent cell aliquots to -80 degrees C.
?. Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins).
-80 °C
?. Remove agar plates (containing the appropriate antibiotic) from storage at 4°C and let warm up to room temperature and then (optional) incubate in 37°C incubator.
37 °C
?. Mix 1μl of DNA (usually 10 pg - 100 ng) into competent cells. Gently mix by flicking the bottom of the tube with your finger a few times. Incubate the competent cell/DNA mixture on ice for 20-30 mins. ~
10 ng
100 ng
?. Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 90 secs.
42 °C
?. Put the tubes back on ice for 2 min.
?. Add 600μl LB media (without antibiotic) to the bacteria and grow in 37°C shaking incubator for 45 min.
600 µl
37 °C
?. The bacterial liquid was centrifuged at 3500rpm for 3 minutes, 400 microliters of supernatant was discarded, and the bacterial liquid was suspended again.
?. Plate the transformation onto a LB agar plate containing the appropriate antibiotic.
?. Incubate plates at 37°C overnight.
37 °C
TITLE: Assay for PhosphoRab activation of LRRK2 Kinase
AUTHORS: Claire Y Chiang, Suzanne R Pfeffer

[DESCRIPTION]
MST kinase phosphorylates Rab proteins at the same site as LRRK2 and has been used to phosphorylate Rab8A and Rab10 quantitatively.  This protocol includes a method to produce phosphoRab8A protein and remove as much contaminating MST3 as possible, to enable use of the phosphoRab to test subsequent activation of LRRK2 kinase.  See these references for details on MST3 phosphorylation of Rab GTPases:
 

 

 


[STEPS]
SECTION: A.  Phosphorylate Rab8A and remove MST3 kinase
11. To cleave the HIS tag from MST3, bind GST-PreScission protease (50 µg) to 100 µL glutathione agarose slurry (pre-washed and pelleted) 120 min at 4 °Cin a total volume of 500 µL. Wash the resin 3X with 1 mL reaction buffer.  Add His-MST3 kinase (0.5 mg) and incubate   on a rotator at 4 °C.

SECTION: A.  Phosphorylate Rab8A and remove MST3 kinase
11. Spin beads 3000 rpm, 5 min, 4 °C and collect supernatant, which contains free MST3 and uncleaved His-MST3. Bind supernatant to Nickel-NTA agarose (100 µL of a 50% slurry, prewashed and pelleted) for 120 min at 4 °C to trap uncleaved His-MST3.

SECTION: A.  Phosphorylate Rab8A and remove MST3 kinase
11. Spin beads 3000 rpm, 5 min, 4 °C and collect supernatant, which contains free MST3 without the HIS tag.

SECTION: A.  Phosphorylate Rab8A and remove MST3 kinase
11. Phosphorylate His-Rab8A Q67L full length with free MST3 using a molar ratio of 1:6 (kinase:substrate) at 30 °C120 min in reaction buffer. 

SECTION: A.  Phosphorylate Rab8A and remove MST3 kinase
11. Phosphorylated His-Rab8A is then separated from MST3 by gel filtration using a 24 mL Superdex 75 10/300 column (Cytiva Life Sciences, #17517401). Collect the relevant Rab-containing fractions determined by SDS-PAGE.

SECTION: A.  Phosphorylate Rab8A and remove MST3 kinase
11. Further purify His-Rab8A by binding to Nickel-NTA agarose (100 µL of a 50% slurry, prewashed and pelleted) and elute with 500 millimolar (mM) imidazole (3 X 100 µL) after washing with 60 column volumes of reaction buffer.

SECTION: B.  To measure phosphoRab activation of LRRK2 kinase:
11. Incubate 88 nanomolar (nM) LRRK2 G2019S with 3 micromolar (µM) His-GFP-Rab10 Q68L 1-181 or His-SUMO-Rab10 wild type full length substrate ± 6 micromolar (µM) phosphoRab8A Q67L in reaction buffer in a total volume of 100 µL.

SECTION: B.  To measure phosphoRab activation of LRRK2 kinase:
11. Incubate reaction in a 30 °C water bath and collect 20 µL time points at 0, 10, and 20 minutes. Stop reactions with addition of 5 µL SDS-PAGE sample buffer. Add 200 nanomolar (nM) MLi-2 for control conditions to ensure that pRab10 detected is due to LRRK2 activity.

SECTION: B.  To measure phosphoRab activation of LRRK2 kinase:
11. Analyze samples by SDS-PAGE and immunoblot for phosphoRab10; image blot with Li-COR and quantify bands using ImageJ (see below for more details).
TITLE: Protocolo para PFA 4%
AUTHORS: Hector Silva

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Solução para fixar tecidos</div></div>

[STEPS]
?. Aquecer a Água destilada até 70°C, no misturador, colocar a “pulga” (imã) e liga-lo, acrescentando aos poucos o PFA
?. Jogar duas pedrinhas ou mais de NaOH até a mistura ficar clara e homogênea. Por último, acrescentar a solução de PB 0,2M
TITLE: Karnovsky’s Modified Fixative
AUTHORS: Mason Mackey, Mark Ellisman, Marianne Martone

[DESCRIPTION]
Fixative for mice and rat tissues to be used during perfusion and immersion fixation. 

[BEFORE_START]
Make sure heating plate for dissolving paraformaldehyde is between  60-70° C. 

[GUIDELINES]
Preparing Fix solution (2% paraformaldehyde and 2.5% glutaraldehyde)

For 100 mL of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.15M cacodylate buffer solution at 7.4 pH.


[STEPS]
1. Add 2.0 grams paraformaldehyde to a 100mL Erlenmeyer flask.
2. Add 35mL of DDH2O to 100mL to a Erlenmeyer flask.
3. Heat paraformaldehyde in DDH2O to 60-70ºC.  Use stir bar to mix.
4. Add 2-3 drops 1N NaOH.
5. Filter solution with #1 Whatman filter into a 100mL graduated cylinder. 
6. Add 10.0 ml of 25% glutaraldehyde.
7. Dilute with DDH2O to a total volume 49.0 ml.
8. Add 1.0 ml of 0.204 M CaCl2-2H2O.
9. Add 50 ml 0.3M cacodylate buffer stock solution at pH 7.4.
10. Volume should be 100 ml. Transfer fixative into a 100mL Erlenmeyer flask. 
11. Keep in bath water 35ºC.
TITLE: scRNA-seq Data Processing (Cell Ranger, v6.0.0)
AUTHORS: molmer  , Martin  Lotz, Padmaja  Natarajan, Steven  Head

[DESCRIPTION]
scRNA-seq data are analyzed using Cell Ranger from 10X Genomics.

[STEPS]
1. The "cellranger mkfastq" pipeline is used to demultiplex the raw Illumina base call files (BCL) into fastq format for further analysis. This pipeline uses a custom-built wrapper around Illumina’s bcl2fastq to demultiplex raw BCL files.
3. Differentially expressed genes identified between clusters of interest are further explored for pathways they impact using softwares such as Advaita's iPathwayGuide and Gene Set Enrichment Analysis (GSEA).
2.1. Reads are aligned to the GRCh38 (GENCODE v32/Ensembl 98) human reference genome using STAR. STAR performs splicing-aware alignment of reads to the genome. Aligned reads are then bucketed into exonic and intronic regions using a reference GTF file.
2. The "cellranger count" pipeline is then used to align reads to a reference genome, filter reads, count cellular barcodes and count UMIs.
2.2. The final "cellranger count" output yields gene expression matrices for each sample that contain the UMI counts per gene, per cell barcode. The results also include summary files with summary metrics describing sequencing quality along with secondary analysis output that includes PCA, t-SNE and UMAP plots; details on grouping of cells with similar expression profiles (clustering) and features differentially expressed in each cluster relative to all other clusters.  The output ".cloupe" files are used for visualization and analysis in Loupe Browser.
TITLE: DAB-quant
AUTHORS: Sneh  Patel,   Sara Fridovich-Keil,   Shauna Rasmussen, and  Judith L Fridovich-Keil

[DESCRIPTION]
Here we describe DAB-quant, a new system that facilitates quantitation of large numbers of scanned tissue slides stained via immunohistochemistry with 3,3′-Diaminobenzidine (DAB). The python code, instructions, license, and a link to example scans for analysis are all available at: 
https://github.com/sarafridov/DAB-quant

[STEPS]
1. Here we provide DAB-quant, a new system that facilitates quantitation of large numbers of scanned tissue slides stained via immunohistochemistry with 3,3′-Diaminobenzidine (DAB). The python code, instructions, license, and a link to example scans for analysis are all available at:
 https://github.com/sarafridov/DAB-quant
TITLE: Neuropathy Phentoyping Protocols - Immunofluorescence Method for 8DG Localizaiton
AUTHORS: Eva Feldman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block"><span style = "font-weight:bold;">Phenotyping of Rodents for the Presence of Diabetic Neuropathy </span></div><div class = "text-block">In man, the development of diabetic neuropathy is dependent on both the degree of glycemic control and the duration of diabetes. Diabetic neuropathy is a progressive disorder, with signs and symptoms that parallel the loss of nerve fibers over time. Consequently, assessments of neuropathy in mice are not performed at one time point, but are characterized at multiple time points during a 6 month period of diabetes. The degree of diabetes is evaluated in 2 ways: tail blood glucose measured following a 6 hour fast and glycated hemoglobin levels. The initial degree of neuropathy is screened using the methods discussed below. Detailed measures of neuropathy are employed when the initial screening instruments indicate a profound or unique phenotypic difference. This document contains protocols used by the DiaComp staff to examine and measure diabetic neuropathy at the whole animal, tissue and cellular levels. </div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complication:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/binm4kcn.jpg" /></div><div class = "text-block"><span style = "font-weight:bold;">Reference:</span></div><div class = "text-block"><span style = "font-style:italic;">Yarborough et al, Cancer Res. 56:683-688, 1996</span></div></div>

[STEPS]
?. Deparaffinize slides by soaking in Hemo-D overnight in rocker oven. If using cryosections, thaw on warm plate 10 min, ring with PAP pen.
?. Re-hydrate through EtOH 100% -50% dH²O.
?. Equilibrate sections in Tris. (10 mM, pH 7.5, 1 mM EDTA, 0.4 M NaCl)
?. Incubate with RNase in Tris, 100 µg/ml, 37ºC, 1 hour.
?. Rinse 2 X 10 min Tris.
?. Incubate with Proteinase K 10 µg/ml Tris 22ºC, 7 min.
?. Rinse 2 X 10 min Tris.
?. Incubate in 4 N HCl 22ºC, 7 min.
?. Rinse 2 X 10 min Tris.
?. Rinse 2 X 10 min PBS.
?. Begin normal IHC procedure with the 8DG antibody.
TITLE: Single-nuclei suspensions from primary human esophagus tissue
AUTHORS: Lucy Kimbley, Rachel Parker, Jack Harrington, Rob Walker, Annette Hayden, Jonathan West, Peter Johnson, TIm Underwood, Matthew Rose-Zerilli

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A protocol to dissociate frozen esophagus tissue specimens for single-nuclei transcriptomics.</div></div>

[STEPS]
?. Pre-chill Dounce homogenizer with pestle B, containing 2 ml of ice-cold Nuclei EZ lysis buffer.
?. Remove tissue from storage (-20°C in RNALater-ICE) and hold in a petri dish on wet ice. If not utilizing all of the frozen tissue, sever off the amount of tissue required (using a cold scalpel) and return the remainder to original storage site.
?. Using a cold scalpel cut tissue into very small pieces and transfer to Dounce homogeniser. Do not overload homogeniser with tissue (~30 mg per Dounce at one time).
?. Homogenize for 30 strokes with pestle B (pre-chilled in ice-cold in Nuclei EZ lysis buffer. Tissue should be reduced to ~90% homogenous small particles. [Perform the homogenization on ice, occasionally removing from the ice to check the homogenate].
?. With a 1 mL pipet, transfer homogenate to 15 mL conical centrifuge tube (on ice) and rinse the Dounce homogenizer out with a further 1 mL of ice-cold Nuclei EZ lysis buffer. Take care not to transfer any large particulates of unhomogenized tissue. This is a potential source of activated RNAses that have undergone renaturation. Incubate on ice for 5 minutes.
?. Filter suspension through a 40 μM cell strainer in to a 50 ml conical centrifuge tube.
?. Centrifuge nuclei/tissue suspension at 700 x g at 4°C for 5 minutes.
?. Re-suspend isolated nuclei in 4 ml of ice-cold Nuclei EZ lysis buffer and incubate on ice for a further 5 minutes.
?. Centrifuge nuclei suspension at 500 x g at 4°C for 5 minutes.
?. Re-suspend nuclei in 3 mL NSB and filter through a 30 μM strainer in to a 50 mL conical centrifuge tube.
?. Centrifuge nuclei suspension at 500 x g at 4°C for 5 minutes.
?. Re-suspend nuclei in 1-2 mL NSB and hold on wet ice (4°C).
?. Dilute 10 μL of nuclei suspension 1 in 10 with NSB in to a micro-centrifuge tube and count nuclei using 1:2 dilution with dye (either 10 μl Trypan blue or 10 μl DAPI stain (300 nM in PBS; incubate for 2 minutes with nuclei before counting) added to 10 μl of nuclei suspension) and a haemocytometer (Improved Neubauer).