eamag/protocols-with-steps huggingface dataset:
TITLE: Buprenorphine to Improve HIV Care Engagement and Outcomes (BRAVO) Randomized Trial: Baseline analysis protocol by C. King (P. Korthuis team)
AUTHORS: Caroline King

[STEPS]
?. [Overview]
This protocol is for data analysis as part of the Buprenorphine to Improve HIV Care Engagement and Outcomes (BRAVO) Randomized Trial. The BRAVO trial enrolled patients with HIV and moderate-to-severe opiate use disorder to receive either methadone maintenance therapy (MMT) or buprenorphine/naloxone (BUP/NX) at six Vietnamese HIV clinical research sites between 2015 and 2019.
?. [Study Outcome]
The study outcome is HIV suppression at time of enrollment, quantified as less than 200 copies of HIV-1 RNA virus in lab testing.
?. [Covariates]
Binary and categorical covariates included gender (male/female), greater than 9th grade education (yes/no), currently employed (yes/no), currently married (yes/no), currently on ART (yes/no), Hep B positive (yes/no), Hep C antibody positive (yes/no), positive tuberculosis screen (yes/no), history of 06 rehabilitation (yes/no), and other drug use (methamphetamine or amphetamines/methadone/buprenorphine/daily tobacco use/heavy alcohol use/none). Heavy alcohol use will be defined using the AUDIT score. Continuous covariates include mean age and years since HIV diagnosis at the time of diagnosis. Age will be reclassified into 10-year categories during covariate testing to increase usefulness in study results.
?. [Data Analysis]
Primary AnalysisTwo logistical regression models (univariate + age and sex, and univariate + all covariates) will be built to model the relationship of baseline participant characteristics with the binary variable for HIV suppression (HIV-1 RNA
?. [Data Analysis]
Missing DataMissing HIV viral load at baseline will be imputed as “not suppressed”. We anticipate minimal missingness in baseline data among covariates, and will analyze this data using complete case analysis.
?. [Data Analysis]
Model FittingWe will fit the logistic regression using an estimated covariate ratio of 10 events per degree of freedom.All continuous covariates will be evaluated for linearity in the log-odds using Lowess scatter plot (comparing HIV suppression and each continuous covariate, individually). Covariates will be examined for multi-collinearity. If covariates are colinear (p>0.80), the research team has decided to include covariates with more variability among participants; where variability is similar, the research team will consult the clinical team for guidance.
?. [Data Analysis]
Model testingWe will carry out Hosmer-Lemeshow tests for regression models to ensure the models are well-fit, using an alpha level of 0.05.
?. [Data Analysis]
Sensitivity analysisOne sensitivity analysis for the regression model will be conducted. Influential observations will be identified using Pregibon’s Delta-Beta statistic; observations with a Delta-Beta statistic greater than 0.20 were removed. Significant sensitivity analysis results will be reported alongside primary analysis results.
?. [Technology]
All data will be analyzed using Stata IC Version 15.
TITLE: Capped RNA Synthesis (E2050)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
The kit formulation allows for efficient capped RNA synthesis using cap analog (ARCA).

[BEFORE_START]
We strongly recommend wearing gloves and using nuclease-free tubes and reagents to avoid RNase contamination. Reactions are typically 20 μl but can be scaled up as needed. Reactions should be assembled in nuclease-free microfuge tubes or PCR strip tubes.

The kit formulation allows for efficient capped RNA synthesis using cap analog (ARCA). The recommended ratio of cap analog to GTP is 4:1. Increasing the ratio of cap analog to GTP will increase the proportion of capped RNA transcripts; however, it also significantly decreases the yield of the reaction. Cap analogs are sold separately. Please refer to the companion products section.

[GUIDELINES]
We strongly recommend wearing gloves and using nuclease-free tubes and reagents to avoid RNase contamination. Reactions are typically 20 μl but can be scaled up as needed. Reactions should be assembled in nuclease-free microfuge tubes or PCR strip tubes.

The kit formulation allows for efficient capped RNA synthesis using cap analog (ARCA). The recommended ratio of cap analog to GTP is 4:1. Increasing the ratio of cap analog to GTP will increase the proportion of capped RNA transcripts; however, it also significantly decreases the yield of the reaction. Cap analogs are sold separately. Please refer to the companion products section.

Cap analog (ARCA, NEB #S1411) is supplied in a lyophilized form of 1 µmol per tube. Dissolving it in 25 μl nuclease-free water will yield a concentration of 40 mM.

The yield per reaction is 30–40 μg RNA with approximately 80% capped RNA transcripts. Figure 1 shows the time course of capped RNA synthesis from 1 µg control template. Most reactions will be complete in 1 hour.
 
Figure 1. Capped RNA Synthesis with ARCA
Reactions were incubated at 37°C in a thermocycler. Transcripts were purified by spin columns and quantified on a NanoDrop Spectrophotometer.

[STEPS]
1. Prepare 40 Milimolar (mM).
 

2. Thaw the necessary kit components, mix and pulse-spin in a microfuge to collect solutions to the bottoms of tubes.
3. Assemble the reaction at Room temperature in the following order:  
 

4. Mix thoroughly and pulse-spin.
5. Incubate at 37 °C for 120 min.
 


  

6. Optional step: To remove template DNA, add 2 µL, mix and incubate at 37 °C for 15 min.
7. Proceed with purification of synthesized RNA (we recommend the Monarch RNA Cleanup Kits, NEB #T2040 or #T2050) or analysis of transcription products by gel electrophoresis.
TITLE: Equine parietal bone harvesting
AUTHORS: Lilibeth A Zambrano M, David Kilroy, Arun Kumar, Michael D. Gilchrist, Aisling Ni Anaidh

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is the procedure carried out to etract beam-like specimens from the parietal bone of horse skull.</div></div>

[STEPS]
?. [Braincase defleshing]
In a room at 22°C:- Using a scalpel, carefully remove soft tissue removed from the bone.- Remove the soft organs attached to the inner surface of the bone (brain, cerebellum, dura mater, etc.).- Permanently irrigate the bone with saline solution.
?. [Pre-sampling template]
- With the pencil, draw a square upon the parietal bone of the skull. The dimensions should cover the surface of at least one rectangular sample: 52 mm * 12 mm (taking into account the thickness of the saw). Note: alignment of the sample template should be done according to the direction to test. - The rectangle size will change according to the number of samples to cut.
?. [Sample cut]
- Align the skull so the vertical band saw is perpendicular to the surface to cut. - Carefully cut the individual sample following the pattern previously drawn, letting the saw go all the way through the thickness.- Permanently irrigate the bone with saline solution
?. [Sample storage]
- Wrap individual samples in gauze sodden with saline solution.- Store at 4°C.
?. [Braincase extraction]
In a room at 22°C:- Carefully place the horse head in the vertical band saw - Cut the section that encloses the braincase: from the occipital bone to the beginning of the frontal bone.- Permanently irrigate the bone with saline solution.At least two people should be involved in this part of the process
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>For RNA extraction, the tissues were ground and the total RNA was isolated using NucleoSpin® RNA columns (Macherey-Nagel; Düren, Germany) according to the manufacturer’s instructions. RNA was quantified using NanoDrop® spectrophotometer ND-1000 (Thermo Scientific Fisher; Waltham, MA, USA). Total RNA was reverse-transcribed into cDNA with a High Capacity Reverse-Transcription kit (iScript<sup>TM</sup> cDNA synthesis KIT; Bio-Rad, Hercules, CA, USA). Real-time PCR detection was performed with a SYBR Green supermix (Bio-Rad, Hercules, CA, USA). Gene specific primers were designed and checked using the BLAST algorithm. Gene expression was normalized using the expression of GAPDH, β-Actin and RPS13.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Mechanical dissociation of human spleen tissue to single cells for use in 10X chromium scRNA-Seq preparation. </p>

[STEPS]
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TITLE: Phosphoglycerate kinase is a central leverage point in Parkinson’s Disease driven neuronal metabolic deficits.
AUTHORS: Alexandros C Kokotos, ama, Santiago Unda, Myung Soo Ko, Daehun Park, David Eliezer, Michael G. Kaplitt, Pietro De Camilli, Tim Ryan

[DESCRIPTION]
The current collection contains all experimental protocols associated with the study "Phosphoglycerate kinase is a central leverage point in Parkinson’s Disease driven neuronal metabolic deficits".

[STEPS]
TITLE: Intraperitoneal Glucose Tolerance Testing (IPGTT)
AUTHORS: Donald McClain

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">This is the standard protocol for most routine glucose tolerance testing. It is performed on awake mice, fasted for 6 hr.</div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complications:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bie54kcn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bie64kcn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bie74kcn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bie84kcn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bie94kcn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bifa4kcn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bifb4kcn.jpg" /></div></div>

[STEPS]
?. Mice are fasted for six hours by removal to a clean cage without food at the end of their dark (feeding) cycle.
?. Mice are weighed.
?. After 6h of fasting, a fasting glucose level is obtained from venous blood from a small tail clip.
?. 1 mg/g body glucose is injected intraperitoneally.
?. Blood glucose values are obtained at 5, 15 30, 60, and 120 min.
?. If desired, blood may also be obtained for insulin assay (~50 µl) at any time point.
TITLE: Treatment regimens used in the management of Helicobacter pylori in Colombia
AUTHORS: Luis Valladales-REstrepo, Yessenia Correa-Sanchez, Brayan Stiven Aristizábal-Carmona, Jorge        Machado Alba

[DESCRIPTION]
Background:Helicobacter pylori infection can cause gastritis, gastric ulcers, duodenal ulcers and gastric cancer. Its treatment involves different medications, but resistance to these treatments is increasing. It is currently considered a public health problem. The objective was to identify H. pylori eradication regimens by age group, year of treatment and geographical region of Colombia.
Materials and methods: This was a cross-sectional study that identified the H. pylori eradication regimens used for patients treated in outpatient consultations over a 6-year period based on a medication dispensing database of 8.5 million people affiliated with the Colombian Health Care System. Sociodemographic and pharmacological variables were considered. A descriptive analysis was performed.
Results: A total of 12,011 patients with a diagnosis of acid-peptic disease and H. pylori infection were identified. They had a median age of 49.9 years, and 65.5% were women, and they had undergone 12,426 eradication treatment regimens. Of these, 98.0% used a proton pump inhibitor (PPI), and 91.1% used amoxicillin. A total of 56.1% of the regimens were considered adequate; of these, 42.0% corresponded to the combination of PPI, amoxicillin and clarithromycin. This regimen predominated between 2015 and 2017 for all age groups and in all geographic regions except the Amazon-Orinoquía-Eastern region. Starting in 2018, the PPI, amoxicillin and metronidazole regimens predominated.
Conclusions: The management of H. pylori infection in the majority of patients is heterogeneous and inconsistent with current recommendations based on evidence of antimicrobial resistance.

[STEPS]
TITLE: 10xV3 Genomics Sample Processing Protocol
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol allows for rapid generation of 3’ transcriptomic-NGS-ready- single-cell-libraries from pools of cells.</div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p><strong>REAGENT PREPARATION</strong></p>
<p> </p>
<p>★<strong> Laird’s buffer (100ml) </strong>(adjust pH to 8,5) [store at room temperature]</p>
<p> </p>
<p>Tris                           1,21 g</p>
<p>EDTA-NA<sub>2              </sub>0,19 g</p>
<p>NaCl                        1,17 g</p>
<p>SDS                           0,2% (= 1 ml if 20% SDS stock is used)</p>
<p>ddH<sub>2</sub>O                    99 ml (depends of SDS’s volume)</p>
<p> </p>
<p>★ <strong>Proteinase K (20mg/ml): </strong>Dilute 10 mg of proteinase K in 0,5 ml of ddH<sub>2</sub>O. [store at -20ºC]</p>
<p> </p>
<p>★ <strong>Phenol: </strong>stock concentration</p>
<p> </p>
<p>★ <strong>P/C/I, Phenol-Chloroform-Isoamyl (25:24:1)</strong> [store at 4ºC]</p>
<p> </p>
<p>To prepare a 250 ml solution:</p>
<p>Phenol                                    125 ml</p>
<p>Chloroform         120 ml</p>
<p>Isoamyl                 5 ml</p>
<p> </p>
<p>★ <strong>C/I, Chloroform-Isoamyl (24:1)</strong> [store at 4ºC]</p>
<p> </p>
<p>To prepare a 250 ml solution:</p>
<p>Chloroform         240 ml</p>
<p>Isoamyl                                  10 ml</p>
<p> </p>
<p>★ <strong>NaCl 5M</strong>: 29,24 g of NaCl is dissolved in H<sub>2</sub>O up to 100 ml. Use it within 1 month.</p>
<p>Alternatively,<strong> NaAc 3M</strong>: 40,8 g of NaAc is dissolved in H<sub>2</sub>O up to 100 ml. [store at RT]</p>
<p> </p>
<p>★ <strong>Ethanol 100% </strong>or alternatively: propanol. [store at room temperature]</p>
<p><strong> </strong></p>
<p>★ <strong>Ethanol 70%:</strong> To prepare 50 ml: 36,5 ml Ethanol 100% + 13,5 ml ddH<sub>2</sub>O. [store at -20ºC]</p>

[STEPS]
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TITLE: Emir&#39;s methanol fixation IF protocol
AUTHORS: Emir  Bora Akmeriç

[DESCRIPTION]
Methanol fixation protocol for immunofluorescence, should hopefully work for Claudin-5 staining.

[STEPS]
SECTION: Fixation
1. Bring methanol to -20C and prepare compartment in cell culture freezer to quickly transfer slides/dishes. Get a box of ice-20 °C   

SECTION: Fixation
2. Incubate sample in methanol for 12 minutes. 
SECTION: Fixation
3. Wash samples with PBS 3 times on ice
SECTION: Fixation
4. Incubate samples with PBS on ice for 5 minutes, repeat 3 times

SECTION: Fixation
5. Store samples in the fridge until further use4 °C 

SECTION: Blocking/Staining
6. Block for 1hr in 5% normal goat serum diluted in PBS
SECTION: Blocking/Staining
8. Incubate primary antibody diluted in blocking buffer overnight at 4C.
SECTION: Blocking/Staining
9. Wash samples with PBS 3 times

SECTION: Blocking/Staining
10. Incubate samples with PBS for 5 minutes, repeat 3 times
SECTION: Blocking/Staining
11. Incubate with secondary antibody diluted to 1:1000 in blocking buffer for 2h
SECTION: Blocking/Staining
7. Make primary antibody mix for the next step in the meantime
SECTION: Mounting
12. Wash samples with PBS 3 times

SECTION: Mounting
13. Incubate samples with PBS for 5 minutes, repeat 3 times
SECTION: Mounting
14. Add 1:100 DAPI diluted in PBS, incubate for 10 minutes
SECTION: Mounting
15. Wash samples with PBS 2 times

SECTION: Mounting
16. Add mounting media, probably PBS in this case or mowiol mixed with 10% dabco
SECTION: Blocking/Staining
14. Incubate in 1:100 DAPI diluted in PBS for 10 mins at RT
SECTION: Blocking/Staining
15. Wash 2x with PBS for 5 mins
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p> This is a protocol especielly for the isolation of Viral Like Particles (VLP) from the tissues of Molluscs, such as abalone, oyster, clam,  scallop and so on. This maybe also applicable for other animal tissues, such as shrimp and fish, based on the principle of the protocol. But we havn't verified this idea yet.</p>
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[GUIDELINES]
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[STEPS]
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TITLE: Generation of membrane tubules by lipid-covered silica beads rolling
AUTHORS: Javier Espadas, Aurelien Roux

[DESCRIPTION]
This protocol explains the high throughput methodology to generate membrane tubules from lipid-covered silica beads.

[STEPS]
SECTION: Protocol
1. Mix of DOPC, DOPS and Atto 647N DOPE at 59.9:40:0.1 mol% respectively in a final volume of 200 µL with chloroform and 0.5 g/L lipid final concentration in a glass vial.
SECTION: Protocol
2. Dry the lipid mixture in the glass vials for 120 min in a vacuum chamber forming the dried lipid films on the bottom of the glass vials.
SECTION: Protocol
3. Add 200 µL of the lipid films hydration buffer A to the glass vial containing the dried lipid films.

SECTION: Protocol
4. Vortex the glass vials until visually seeing complete resuspension of the dried lipid films in the solution (seen by an increase in the turbidity of the lipid solution) forming the multilamellar vesicles (MLVs).
SECTION: Protocol
5. Mix 10 µL of MLVs with 2 µL of silica beads in an Eppendorf.

SECTION: Protocol
6. Deposit 6 drops of 2 µL each containing the mixture of MLVs and silica beads on a parafilm slide placed in the bottom of a petri dish.
SECTION: Protocol
7. Dry the drops for 60 min in the vacuum chamber until the liquid is completely dried.
SECTION: Protocol
8. Stick a microfluidic device on a 1.5 borosilicate coverslip.
SECTION: Protocol
9. Passivate the microfluidic channels by adding 200 µL solution of 2 g/L BSA for 15 min.

SECTION: Protocol
10. Clean the BSA solution by passing 200 µL working buffer solution in each channel 5 times.

SECTION: Protocol
11. Add 200 µL of working buffer to each channel with a final concentration of GFP-LRRK2 of 500 nanomolar (nM).

SECTION: Protocol
12. Pick one of the dried drops and add it to the inlet of the microfluidic device.
SECTION: Protocol
13. Gently tilt the chamber towards you 60 degrees with the inlet in the upper part and the outlet in the lower part, and wait until visually seeing the lipid-covered silica beads moving from the inlet to the outlet openings of the microfluidic device.
SECTION: Protocol
14. Wait until reaching the steady state of protein coverage on the lipid tubules.
TITLE: Changes in Bone Mineral Density and Incidence of Fractures during Two Years of Low Dose Glucocorticoid Treatment for Rheumatoid Arthritis: Protocol for a Systematic Review and Individual Participant Data Meta-Analysis
AUTHORS: Andriko Palmowski

[DESCRIPTION]
Background
Glucocorticoids (GCs) are regularly used drugs in the treatment of rheumatoid arthritis (RA), and lower bone mineral density and fractures are common adverse effects of the treatment with higher GC dosages. However, the effects of low dose (i.e., ≤7.5mg/day) and very low dose (i.e., ≤5mg/day) treatment over longer periods of time (i.e., ≥ 24 months), as often seen in RA, have not been fully elucidated yet.

Objective
To conduct a systematic review and meta-analysis of individual patient data from long-term randomized controlled trials (RCTs) in RA, which compared low dose GCs to a control treatment, in order to investigate the effects on bone mineral density and incidence of fractures (clinical/symptomatic). 

Methods 
We will search the literature to identify published trials which collected data on bone mineral density and/or the incidence of fractures. This will be followed by the acquisition of individual participant data. Included RCTs will be combined to compare bone mineral density and fractures in GC and control groups. To underpin our findings, we will perform additional analyses to identify potential effect modifiers and a sensitivity analysis related to missing data. 

[STEPS]
SECTION: PDF Protocol
1.
TITLE: Cryopreservation of stem cell derived ventral midbrain neural progenitors
AUTHORS: Tyra Fraser, Lachlan Thompson

[DESCRIPTION]
This protocol outlines the cryopreservation procedure for stem cell derived neural progenitors. It can be used for the cryopreservation and long-term storage of ventral midbrain dopamine neuron progenitors at Day 13 or Day 17 of differentiation in liquid nitrogen tanks.  

[BEFORE_START]
Day 17 (D17) VmDA  and day 13 (D13) VmDA progenitors are used in this protocol

[GUIDELINES]
All work is to be conducted in well sterilised laminar flow hoods designated for human iPSC work where possible to minimise contamination

[STEPS]
SECTION: Experimental procedure
1. Take Mr. Frosty‱ out of -80 °Cfreezer and keep at Room temperature .
Label the cryogenic vials with a label machine to include the following information: cell line, clone number, day of differentiation, freezing date and initials. 
Wash cells with PBS -/-
Put 484 µL of Accutase per cm2 of cells. 
Incubate cells at 37 °C4 min 
Whilst cells are incubating place 5 mL of PBS -/- into a 15 mL Falcon tube.
Remove cells from incubator and pipette up and down twice. If cells come off in large clumps place them back into the incubator for 2 min. Repeat this step until cells form small clumps (~5cells).  If cells form small clumps after the first 4-minute incubation move straight to step 8. 
Place cells in the falcon tube prepared earlier (Step 6) with Ri 1:1000.
Spin cells300 x g, 3 min, 4 °C
SECTION: Experimental procedure
2. Aspirate supernatant and flick pellet twice. 
Resuspend in1 mLof NBB27+ Ri (1:1000). 
 Put 10 µLof cell mix into a small Eppendorf tube for cell counting.  
Add 10 µL trypan blue to cells and mix well by pipetting up and down. 
 Transfer 10 µL of cells + Trypan blue mix in haemocytometer.
Count cells in each quadrant.
Calculate total number of cells.

Total cells = Average count of quadrants x Dilution factor x Volume (ml) x 10^4

8. Calculate volume required for desired freezing concentration. We recommend freezing no less than 1x106 cells per vial. 
9.       Spin cells300 x g, 3 min, 4 °C
SECTION: Experimental procedure
3. 1.  Aspirate supernatant and flick pellet twice. 
2. Resuspend in desired amount of Cryostor‱ CS10 for each cryovial (Calculated in Step 8 above). 

IMPORTANT: Be fast to avoid cell death due to toxic Cryostor ‱ CS10.

3. Quickly aliquot1 mLof cells + Cryostor ‱ CS10 into cryovials. 
4. Put the vials in Mr.Frosty and place in the -80°C freezer as fast as possible.
5. 1440 min after freezing, transport the cells on dry ice and store in Liquid Nitrogen for
long-term storage.

IMPORTANT: Do not store cells in the -80 freezer for longer than 1 week.