eamag/protocols-with-steps huggingface dataset:
TITLE: A protocol for rapid western-blot: shorten the time to 1-3 hours
AUTHORS: Zhichao  Zhao  Shenao Biotechnology Company, wang jiang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Western blot (WB) is a classical and powerful tool to detect the level of interested protein from among a mixture of proteins. Since its invention in 1979, WB is a time-consuming technique that usually needs 1 to 2 days to obtain the result. Here, being aware of the time spent by the researcher, we share a rapid protocol that shortens the time up to 1 hour for detecting the internal loading control protein actin (42kDa) when using the HRP-conjugated anti-actin antibody. Additionally, the time for detecting other (21kDa-100kDa) could be shortened to 3 hours when using the second HRP-conjugated antibody. In conclusion, this rapid protocol dramatically improved the efficiency of WB without increasing the experiment cost or losing its high data quality.</div></div>

[STEPS]
TITLE: RNA cleanup with magnetic beads
AUTHORS: Dominik Buchner

[DESCRIPTION]
This protocol describes cleaning up RNA extracts with carboxylated magnetic beads and a PEG-NaCl buffer. It can also be used for volume reduction of a sample or buffer exchange after enzymatic reactions (e.g. DNAse treatment).



[BEFORE_START]
Make sure all buffers are prepared before starting.
For more effortless pipetting let the bead solution adjust to Room temperature
 



[GUIDELINES]
Follow general lab etiquette. Wear gloves to prevent contaminating the samples. Clean the workspace before starting with 80% EtOH. 

[STEPS]
1. Shake the RNA cleanup solution until the beads are homogeneously resuspended

 

2. To 100 µL  add 200 µL in a 1.2 mL Deep Well Storage Plate

3. To bind the RNA to the beads shake at   

 


4. Place the plate on a magnet to pellet the beads for 5 min or until the mixture appears clear

 


5. Discard the supernatant by pipetting
6. With the plate still on the magnet, add 100 µL to each sample

7. Incubate for at least 30 s 

8. Discard the supernatant by pipetting
9.   

10. With the plate still on the magnet, incubate the plate for 5 min  at Room temperature to dry off residuals of wash buffer

11. Add 100 µL to each sample

12.   to elute the RNA from the beads

13. Place the plate on a magnet to pellet the beads for 5 min 

14. Transfer 100 µL of the eluted RNA to a new storage plate. Store at -80 °C
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The unfolding dynamics of the vocal expression of emotions are crucial for the decoding of the emotional state of an individual. In this study, we analyzed how much information is needed to decode a vocally expressed emotion using affect bursts, a gating paradigm, and linear mixed models. We showed that some emotions (fear, anger, disgust) were significantly better recognized at full-duration than others (joy, sadness, neutral). As predicted, recognition improved when greater proportion of the stimuli were presented. Emotions recognition curves for anger and disgust were best described by higher order polynomials (second to third), while fear, sadness, neutral, and joy were best described by linear relationships. Acoustic features were extracted for each stimulus and subjected to a principal component analysis for each emotion. The principal components were successfully used to partially predict the accuracy of recognition (i.e., for anger, a component encompassing acoustic features such as fundamental frequency (f0) and jitter; for joy, pitch and loudness range).  Furthermore, the impact of the principal components on the recognition of anger, disgust, and sadness changed with longer portions being presented. These results support the importance of studying the unfolding conscious recognition of emotional vocalizations to reveal the differential contributions of specific acoustical feature sets over time. It is likely that these effects are due to the relevance of threatening information to the human mind and are related to urgent motor responses when people are exposed to potential threats as compared with emotions where no such urgent response is required (e.g., joy).</p>

[STEPS]
SECTION: Select recordings from the GEneva Multimodal Emotion Portrayals (GEMEP)
?. 
SECTION: Normalized the recordings and remove silences
?. 
SECTION: Create the gates
?. 
SECTION: Generate pseudo-randomized lists of stimuli
?. 
SECTION: Present stimuli to participants and record responses
?. 
SECTION: Group stimuli responses by bins
?. 
SECTION: Computation of Hu Scores
?. 
SECTION: Linear mixed models
?. 
SECTION: Recognition curves fit and polynomial contrasts
?. 
SECTION: Computation of acoustical features
?.
TITLE: Study Protocol for a Mixed Method, Three-Arm Cluster Randomized Comparative Trial Testing the Impact of Culturally Aware Mentoring Interventions on  Research Mentors and Graduate Training Programs
AUTHORS: Ellyssa Eiring, Angela Byars-Winston, Stephanie C. House, You-Geon Lee, Richard McGee, Remi F. Jones, Sylvia Hurtado, Amanda Carrasco

[DESCRIPTION]
Research mentoring relationships are critical for developing the next generation of scientists. However, research mentors have seldom recognized how cultural differences can influence mentoring relationships, particularly for individuals from backgrounds historically underrepresented in the sciences. To address this need, a mentorship education intervention, the Culturally Aware Mentoring (CAM) workshop, was developed. Preliminary studies showed CAM was effective in promoting culturally aware mentoring principles and behaviors. This paper describes the study protocol for a deeper examination of the impacts of three variations of CAM. We use a mixed method, three arm cluster randomized comparative trial design with 33 sites, paired with in-depth case studies at two institutions. The Theory of Planned Behavior and Organizational Learning provide complementary theoretical frameworks to gauge how mentors and their training environments are impacted by CAM. The primary research questions are: 1) What are the short and longer-term impacts of CAM interventions on research mentors’ attitudes and behaviors? 2) What are the broader institutional impacts of CAM on graduate training environments? 3) Why, or why not, do mentors and training environments change? Study sites are biomedical PhD programs at high research universities across the United States. The study targets 600 faculty actively mentoring PhD students and the interventions are delivered virtually and led by trained facilitators. Survey data are collected across the trial and the case studies at four time points: pre-intervention, immediately post-intervention, 6 and 12 months post intervention. Post intervention interviews are conducted with a subsample of participants to examine how and why their mentoring practices changed. The case studies assess organizational changes in those environments following CAM intervention. This study’s results will reveal extensive insights into the research questions, laying the foundation for broader deployment of CAM to the biomedical and larger research training community.

[STEPS]
TITLE: CalculatingGenerationTime
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Calculation of generation times (doubling times)</div></div>

[STEPS]
?. [Steps]
Plot your OD 600 versus time so that you know that you are in EXPONENTIAL PHASE to calculate GENERATION TIMES
?. [Steps]
Grow ON of strain of interest in appropriate media
?. [Steps]
In the morning, check the culture(s) under the scope to verify pure culture. Dilute the ON into the appropriate media.
?. [Steps]
At hourly intervals, record OD 600 nm. Make sure you record the time in minutes! For example, 2 hours is 120 minutes; 3 hours and 30 minutes is 210 minutes, etc.
?. [Steps]
In order to calculate the generation/doubling time, calculate the log of the OD 600 values you have obtained. This will give you negative numbers unless the OD 600 value is above 1.0 (which in that case you are likely NOT in exponential phase).
?. [Steps]
Calculate the slope (K) of the line by selecting points in mid to late exponential phase. From the example above:        Slope = (N - N0)/(T - T0) = K (slope is called K in theses calculations)
?. [Steps]
Calculate the generation time using the following formula:            tg = 0.3 / K = minutes to double            N.B 0.3 is a constant
?. [Steps]
Here are some values as an example:180 (To)0.3576 (No)-0.447291 (T)0.9525 (N)-0.021Slope (K) = (N – N0) / (T – T0) = ((-0.021) - (-0.447) )/ (291-180) = 0.0038tg = 0.3 / K = 0.3 /0.0038tg = 78.3 minute
180 (To)0.3576 (No)-0.447291 (T)0.9525 (N)-0.021
TITLE: SPARC Serotonin 2B Receptor (5-HT2BR) Immunohistochemistry Protocol in Rat Tissues Labeled with Cholera Toxin B-fragment
AUTHORS: Elisa Gonzalez-Rothi, Yasin Seven, Latoya Allen, Marissa Ciesla, Gordon Mitchell

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the immunofluorescent labeling technique used to identify serotonin 2B receptor expression in CtB-labelled phrenic motor neurons and within a defined region of interest surrounding phrenic motor neurons.</div></div>

[STEPS]
?. Day 1: primary antibodies required:5-HT2BR: Mouse anti-5-HT2BR (SantaCruz Biotechnology #C-6; SC-376878)Cholera toxin B-fragment: Goat anti-CT-B (Millipore #227040)
?. Place 40um transverse spinal cord sections into 1xPBS in 12 well cell culture plates
?. 5x washes in  1xPBS for 5 minutes each at room temperature
?. Blocking: place tissues in 5% Normal Horse Serum (NHS) in 1xPBS-Triton (0.1%) +0.5% bovine serum albumin (BSA) for 60 minutes at room temperature
?. Primary Antibody Incubation: Incubate tissues in: 5%NHS in 1xPBS-Triton (0.1%), Mouse anti-5-HT2BR (1:1000), and Goat anti-Ct-B (1:2500) overnight at 4 degrees C
?. Day 2: secondary antibodies required:AlexaFluor 488: donkey anti-goat (Invitrogen, Ref#A11055)AlexaFluor 594: donkey anti-mouse (Invitrogen, Ref#A21203)
?. 5x washes in  1xPBS for 5 minutes each at room temperature
?. Secondary Antibody Incubation: Incubate tissues in: 5%NHS in 1xPBS-Triton (0.1%), donkey anti-goat (1:1000), and donkey anti-mouse (1:1000) for 2 hours at room temperature
?. 5x washes in  1xPBS for 5 minutes each at room temperature
?. Mount tissues on Superfrost Plus microscope slides
?. Allow slides to dry overnight
?. Coverslip with VectaShield Antifade Hard Set Mounting Medium (Cat#:H-1400)
TITLE: Overview of scRNA-seq of Human Knee Meniscus
AUTHORS: molmer, Martin Lotz

[DESCRIPTION]
The overview provides sub-protocols for each step required to complete scRNA-seq from human knee meniscus.

[STEPS]
1. Tissue Acquisition
dx.doi.org/10.17504/protocols.io.6qpvr614zvmk/v1
2. Tissue Quality Control
dx.doi.org/10.17504/protocols.io.n2bvj695nlk5/v1 

3. Single-Cell Isolation
dx.doi.org/10.17504/protocols.io.n2bvj6k7blk5/v1 
4. cDNA Library Preparation
dx.doi.org/10.17504/protocols.io.e6nvwkw32vmk/v1
5. Data Processing and Quality Control
 dx.doi.org/10.17504/protocols.io.j8nlkk3q6l5r/v1
TITLE: Sistema de categorías para el estudio de la eficacia escolar
AUTHORS: Carla Ortiz-de-Villate, Javier Rodríguez-Santero, Juan Jesus Torres-Gordillo

[DESCRIPTION]
Sistema de categorías para el estudio de la eficacia escolar.  Se ha calculado su confiabilidad y concordancia entre investigadores en nuestra investigación con un alfa de Krippendorff alto (α=0.807).

[STEPS]
TITLE: CUT and RUN
AUTHORS: anita.adami

[DESCRIPTION]
This protocols describe how to perform CUT&RUN on human brain tissue (frozen)

[STEPS]
SECTION: Sample extraction
1. Flash-freeze postmortem brain tissue 
SECTION: Sample extraction
2. Sample 50 mg - 100 mg from human brain tissue and store at -80 °C until use

SECTION: CUT&amp;RUN
3. Activate ConA-coat magnetic beads (Epicypher) by washing twice in bead binding buffer [20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl, 1 mM MnCl2]. Place on ice until use. 
SECTION: CUT&amp;RUN
4. Isolate nuclei from frozen tissue after incubating with Recombinant Alexa Fluor® 488 Anti-NeuN antibody [EPR12763] - Neuronal Marker (ab190195) at a concentration of 1:500 for 30 minutes on ice.
Run nuclei through the FACS at 4 °C with low flowrate using a 100 mm nozzle and isolate 300.000 nuclei Alexa Fluor – 488 positive nuclei. 

SECTION: CUT&amp;RUN
5. Pellet the sorted nuclei at 1,300 x g for 15 min and resuspend in 1 mL of ice-cold nuclear wash buffer (20 mM HEPES, 150 mM NaCl, 0.5 mM spermidine, 1x cOmplete protease inhibitors, 0.1% BSA) and 10 µL per antibody treatment of ConA-coated magnetic beads (Epicypher) added with gentle vortexing (Pipette tips for transferring nuclei were pre-coated with 1% BSA). 

SECTION: CUT&amp;RUN
6. Bind nuclei to beads for 10 min at RT with gentle rotation, and then split bead-bound nuclei into three equal volumes (corresponding to IgG control, H3K4me3 and H3K9me3 treatments). 

SECTION: CUT&amp;RUN
7. Remove wash buffer and resuspend nuclei in 100 µL cold nuclear antibody buffer (20 mM HEPES pH 7.5, 0.15 M NaCl, 0.5 mM Spermidine, 1x Roche complete protease inhibitors, 0.02% w/v digitonin, 0.1% BSA, 2 mM EDTA) containing primary antibody at 1:50 dilution and incubate at 4 °C 10 min with gentle shaking. 

SECTION: CUT&amp;RUN
8. Wash nuclei thoroughly with nuclear digitonin wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1x Roche cOmplete protease inhibitors, 0.02% digitonin, 0.1% BSA) on the magnetic stand. 
SECTION: CUT&amp;RUN
9. After the final wash, add pA-MNase in nuclear digitonin wash buffer and incubate with the nuclei at 4 °C for 60 min. Wash nuclei twice, resuspend in 100 µL digitonin buffer, and chill to 0 °C -2 °C in a metal block sitting in wet ice. 

SECTION: CUT&amp;RUN
10. Stimulate genome cleavage by addition of 2 mM CaCl2 at 0 ºC for 30 min. Quench the reaction by additing 100 µL 2x stop buffer (0.35 M NaCl, 20 mM EDTA, 4 mM EGTA, 0.02% digitonin, 50 ng/µL glycogen, 50 ng/µL RNase A, 10 fg/µL yeast spike-in DNA) and vortex. 

SECTION: CUT&amp;RUN
11. Incubate 30 min at 37 °C to release genomic fragments. Place bead-bound nuclei on the magnet stand and purify fragments from the supernatant using a NucleoSpin clean-up kit (Macherey-Bagel). 

SECTION: Sequencing
12. Prepare Illumina sequencing libraries using the Hyperprep kit (KAPA) with unique dual-indexed adapters (KAPA).
TITLE: Fruit fly potato culture media
AUTHORS: Jorge Frias, Nelson Simões

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Potato-based medium for culture of Drosophila melanogaster. </div></div>

[STEPS]
?. Dissolve all following ingredients in 1.1 L of distilled water:-12.9 g Dried yeast;-1 g White sugar;-40 g Instant mashed potato flakes;-10 g Agar;-0.875 g Ascorbic acid;
?. Boil everything for 15 minutes.
?. Let it cool down to 60ºC and add 8.5 mL of 10% Nipagin solution.
?. Mix well and fill about 1/4 of your preferred fruit fly vials or bottles with the medium.
?. On a clean bench hood, let the medium solidify in the vials with the cap open to avoid water condensation and store at 4ºC for further usage.
TITLE: Kinase-activity tagged (KAT) western blotting
AUTHORS: Masumi Eto, Shuichi Katsuki, Yoshinori Tanaka, Kosuke Takeya

[STEPS]
?. [PROCEDURE]
Experiment is conducted at room temperature unless noted.
?. [PROCEDURE]
Preparation of Laemmli Gel and Laemmli samples using a conventional method.
?. [PROCEDURE]
Running the SDS-PAGE with the samples (10-50µg of total lysates) and colored Mw marker and run electrophoresis.
?. [PROCEDURE]
Preparation of the blotting using PVDF membrane by a standard western blotting protocol.
?. [PROCEDURE]
Washing the membrane for 5 min with 10mL of Buffered 2-propanol.
?. [PROCEDURE]
Denaturation for 15min with 10 ml of 6 M Gu-HCl buffer.
?. [PROCEDURE]
Serial incubation each for 10 min with 10 mL of 3 M Gu-HCl buffer and then 10 mL of 0.1 M Gu-HCl buffer.
?. [PROCEDURE]
Washing for 3x 5min with 10 ml of Renaturation buffer.
?. [PROCEDURE]
Place the membrane in Hybri-bag
?. [PROCEDURE]
Incubation overnight with 2 ml of 0.2mg/mL substrate protein in Renaturation buffer at 4°C.
?. [PROCEDURE]
Rinse with 10 ml of Phosphorylation Buffer.
?. [PROCEDURE]
Incubation for 1h with 2 mL of Phosphorylation Buffer including 1mM ATP using 37°C shaker.
?. [PROCEDURE]
Rinse with PBS.
?. [PROCEDURE]
Incubation for 20 min with 10 mL of 10 % buffered formalin.
?. [PROCEDURE]
Rinse with PBS.
?. [PROCEDURE]
Quenching for 10 min with 10 mL of 0.1 M glycine in PBS.
?. [PROCEDURE]
Blocking for 30 min with Blocking solution.
?. [PROCEDURE]
Washing for 5 min x4 with 20 mL of Washing solution.
?. [PROCEDURE]
Incubation for overnight at 4 °C with primary antibody diluted with Antibody dilution solution.
?. [PROCEDURE]
Washing for 5 min x4 with 20 mL of Washing solution.
?. [PROCEDURE]
Incubation for 45 min with secondary antibody conjugated with HRP (diluted at 1:5,000 with Antibody dilution solution)
?. [PROCEDURE]
Washing for 5 min x4 with 20 mL of Washing solution.
?. [PROCEDURE]
Incubation for 5 min with 2 mL of ECL solution.
?. [PROCEDURE]
Imaging using chemiluminescence imager
?. [PROCEDURE]
Rinse the blot with distilled H2O twice
?. [PROCEDURE]
Staining the blot with Coomassie Brilliant Blue solution or others.
?. [PROCEDURE]
TroubleshootingFor the first assay, a negative control blot, such as no ATP treatment, is highly recommended.Synthetic peptides conjugated with a carrier protein may be used as substrates.Renaturation efficiency may vary among kinases.
TITLE: Proteomic mapping of ER-PM junctions in living HEK293 cells
AUTHORS: Lian He, Yue-He Ding, Peng Tan, Ji Jing, Meng-Qiu Dong, Yubin Zhou

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">Specialized junctional sites that connect the plasma membrane(PM) and endoplasmic reticulum(ER) are intimately involved in controlling lipid metabolism and calcium signaling in mammalian cells. Store operated calcium entry mediated by dynamic STIM1-ORAI1 coupling constitutes one of the most well-established molecular events occurring at ER-PM junctions, but the protein composition at this particular subcellular compartment remain poorly defined. Using an in situ spatially-restricted biotin-labeling coupled with mass spectrometry, we mapped the proteome of intact ER-PM junctions in living cells without disrupting their architectural integrity. Our approaches lead to the discovery of >70 candidate proteins at ER-PM junctions, with the majority falling into the categories of ER/PM-resident proteins, cytoskeletal components, and proteins functioning in intracellular membrane trafficking or post-translational modifications. Although the current protocol is limited to ER-PM junctions, the methods described herein can be readily extended to study other types of inter-membrane appositions in various types of cells.</div></div></div>

[STEPS]
TITLE: Iodine-starch sweating assay
AUTHORS: Fanglin Lu

[DESCRIPTION]
This protocol outlines the procedure for assessing the sudomotor function in mice under restraint stress and under systemic pilocarpine stimulation. Both two kinds of sweating assays were based on the iodine-starch reaction. 

[BEFORE_START]
Check if the restraint tube is intact.
Prepare 100% starch–castor oil (1 g starch per 1 mL castor oil)
Prepare pilocarpine (diluted in 150 µL 0.9% saline).

[GUIDELINES]
Please follow the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guideline.
It is favorable to not perform these two different kinds of sweating assays on the same day.

[STEPS]
SECTION: Restraint stress-induced sweating
1. The mouse is immobilized in a self-made restraint tube.   

SECTION: Restraint stress-induced sweating
2. Apply 10% povidone–iodine solution to the plantar surface of bilateral hind paws with a cotton bud. Once dry, coat the skin surface with a suspension of 100% starch–castor oil.  

SECTION: Restraint stress-induced sweating
3. Changes on footpads are video-recorded for 3 min. The number of dark spots was counted for each paw.
 

SECTION: Pilocarpine-induced sweating
4. The mouse is placed in the supine position under sevoflurane anesthesia (4 volume% in 1 L/min oxygen). 
 

SECTION: Pilocarpine-induced sweating
5. Apply 10% povidone–iodine solution to the plantar surface of bilateral hind paws with a cotton bud. Once dry, coat the skin surface with a suspension of 100% starch–castor oil.  

SECTION: Pilocarpine-induced sweating
6. Inject 100 µg pilocarpine hydrochloride intraperitoneally.150 µL 

SECTION: Pilocarpine-induced sweating
7. Changes on footpads are video-recorded for 5 min. The number of dark spots was counted for each paw.
TITLE: Counting Cells with a Hemocytometer
AUTHORS: JCPrice 

[STEPS]
?. Clean hemocytometer and cover slip
?. Dilute the resuspended cells 1:4 in fresh complete mediaOptional: Instead of media stain suspended cells with Trypan Blue (100 uL of cells 400 dye).  The digital microscopes in C230 don't usually require the Trypan Blue stain
?. Fill hemocytometer with 10 ul each side cell suspension, on both sides. Filling only one side will cause inaccuracies in the volumes and therefore the calculations.
?. Using a 10x microscope, focus on the grid lines then find the first 16 block (4x4) grid in the upper left quadrant of the hemocytometer
?. Count the number of cells in the 16 block grids found in each corner of the Hemocytometer counting only cells fully within the grid as discussed in the attached file
?. Record cell numbers and count other quadrants
?. To calculate the number of viable cells/mL:Take the average cell count from each of the sets of 16 corner squares (1mm2 4x4 grid).Multiply by 10,000 (10^4).Multiply by 5 to correct for the 1:5 dilution from the media/Trypan Blue addition.The final value is the number of viable cells/mL in the original cell suspension.
?. In the biosafety cabinet, clean the hemocytometer and the coverslip with 70% ethanol.
TITLE: Double Digestion
AUTHORS: Georgina Elisa Diego Macías, Ana Belem García González, Irán Alessandra Chaparro Rodríguez, Jair Alexis Gardea Sáenz

[DESCRIPTION]
Protocol for a Double Digestion with EcoRI & PstI

[STEPS]
2. Add2 µL of 10X Buffer (Anza)

1. Add the following material in a Eppendorf tube of 0.2 µL

3. 1 µL of EcoRI (Anza)

4. 1 µL of PstI (Anza)

5. Add the necessary uL of DNA for a concentration of25 ng

6. Fill with H2Oultra pure up to 20 µL

7. Incubate for600 min at 37°C in a thermocycler