eamag/protocols-with-steps huggingface dataset:
TITLE: Induction of aggregation in alpha-synuclein-expressing cells by treatment with preformed fibrils (PFFs)
AUTHORS: Cole S Sitron, Victoria A Trinkaus, F Ulrich Hartl

[DESCRIPTION]
This protocol details how to efficiently produce alpha-synuclein aggregates in cells by templating the misfolding of intracellular alpha-synuclein through treatment with preformed fibrils of alpha-synuclein (PFFs).


[STEPS]
SECTION: Induction of alpha synuclein aggregation
1. Seed cells into plates such that they are ~25% confluent the following day. 

 

SECTION: Induction of alpha synuclein aggregation
2. On the following day, warm PBS and OptiMEM to 37 °C, thaw an aliquot of PFFs, and (if using) allow lipofectamine to reach Room temperature. 

 

SECTION: Induction of alpha synuclein aggregation
3. Dilute the thawed PFFs 1:20 into an eppendorf containing PBS. 

 

SECTION: Induction of alpha synuclein aggregation
4. Sonicate the PFFs in the Bioruptor Plus on high for 25 cycles of 5 s on and 5 s off at 4 °C.

 

SECTION: Induction of alpha synuclein aggregation
5. Make a master mix of PFF and PBS solutions.
SECTION: Induction of alpha synuclein aggregation
5.1. 1 µL of sonicated & diluted PFFs should be added per 10 mm2 of plate area (5 ug alpha-synuclein/10 mm2).

 

SECTION: Induction of alpha synuclein aggregation
5.2. Master mixes should contain a ratio of 50 µL OptiMEM : 20 µL sonicated & diluted PFFs (or PBS as a control) : 3 µL of lipofectamine 3000.

 

SECTION: Induction of alpha synuclein aggregation
5.3. If using lipofectamine, first incubate the lipofectamine 3000 in OptiMEM for 5 min before adding PFFs or PBS. Gently vortex upon addition of lipofectamine 3000.

SECTION: Induction of alpha synuclein aggregation
6. After addition of PFFs/PBS, gently vortex master mix solutions and incubate for 10 min at Room temperature.

SECTION: Induction of alpha synuclein aggregation
7. Add master mix solutions dropwise to cells, gently vortexing before adding to each well.
TITLE: Generation of induced pluripotent stem cells and gene correction
AUTHORS: michela.deleidi

[DESCRIPTION]
iPSC generation and gene correction (CRISPR-CAS9) protocol

[STEPS]
SECTION: Generation of induced pluripotent stem cells and gene correction
1. Skin fibroblasts were reprogrammed by nucleofection with pCXLE- hOct3/4 (RRID:Addgene_27076), pCXLE-hSK (RRID:Addgene_27078),using the Amaxa nucleofection kit for human dermal fibroblasts (Lonza, VPD-100) and program P-022 of the Nucleofector 2b (Lonza).
SECTION: Generation of induced pluripotent stem cells and gene correction
2. Nucleofected fibroblasts were plated in six-well plates coated with Matrigel (Corning)
in DMEM supplemented with 10% FBS (Gibco) and 1% GlutaMAX Supplement (Gibco).
SECTION: Generation of induced pluripotent stem cells and gene correction
3. The following day, the medium was changed to DMEM+/+ (DMEM with 10% FBS and 1% GlutaMAX Supplement and 1% Pen/Strep (Millipore)) supplemented with 2 ng/ml recombinant
basic human fibroblast growth factor (FGF2, Peprotech).
SECTION: Generation of induced pluripotent stem cells and gene correction
4. On day 3 or4 post nucleofection, the medium was changed to E8 medium com-
posed of DMEM F12 with HEPES (Gibco), 128 ng/ml ascorbic acid (Sigma
–Aldrich), 1x insulin-transferrin-selenium (Thermo Fisher
Scientific), 10 ng/mL FGF2 (Peprotech), 500 ng/ml heparin (Sigma-Aldrich), and 2 ng/ml TGFβ1 (Peprotech). E8 medium was supplemented with 100 μm sodium butyrate and 0.1% Pen/Strep.
SECTION: Generation of induced pluripotent stem cells and gene correction
5. Colonies started to appear from day 14 onward.
SECTION: Generation of induced pluripotent stem cells and gene correction
6.  Induced pluripotent stem cells (iPSCs) were cultured on Vitronectin XF (StemCell Technologies) in E8 medium.
SECTION: Generation of induced pluripotent stem cells and gene correction
7. Gene correction for the L444P mutation was performed as previously described in (Schöndorf, D. C. et al., 2014)
SECTION: Generation of induced pluripotent stem cells and gene correction
8. One hour before nucleofection, 10μM Rockinhibitor was added to the iPSC medium. About 240 nM crRNA (IDT): Atto550-labeled tracrRNA (IDT) duplex was complexed with 124μM Cas9 to form the ribonucleoprotein complex (RNP complex).
SECTION: Generation of induced pluripotent stem cells and gene correction
9. iPSCs(1.6 × 10^6) were nucleofected with the RNP complex and 16μg of ssODN using 100μl of Ingenio nucleofection solution (Mirus) with program B-016 of Nucleofector 2b.
SECTION: Generation of induced pluripotent stem cells and gene correction
10. Following nucleofection, the cells were FACSsorted for Atto550-positive cells using a FACS Aria II with a 100-μmnozzle. After sorting, 1 × 10^4 cells were plated per 10-cm dish.
SECTION: Generation of induced pluripotent stem cells and gene correction
11. Colonies were picked and sequenced by Sanger sequencing.
TITLE: Protocol for "Thriving during COVID-19: Predictors of Psychological Well-Being and Ways of Coping”
AUTHORS: dguess 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">COVID-19 has led to global dramatic shifts in daily life. Following the biopsychosocial model of health, the goal of the current study was to predict people’s psychological well-being (PWB) during the initial lockdown phase of the pandemic and to investigate which coping strategies were most common among people with low and high PWB. Participants were 938 volunteers in the United States who responded to an online survey during pre-peak April 2020. The main findings were that all three groups of variables, biological, psychological, and socio-economic, significantly contributed to PWB explaining 53% variance, with social loneliness and sense of agency being the strongest predictors. PWB was significantly predicted by physical health (not gender nor age); by spirituality, emotional loneliness, and sense of agency; by social loneliness, and employment security (not income, nor neighborhood safety, nor hrs. spent on social media). Comparing the coping strategies of participants, results show more intentional coping in the high-PWB group and more passive coping in the low-PWB group. With this unprecedented pandemic, the findings highlight that actively and intentionally attending to one’s PWB by using strategies that own one’s control and being an agent for physical, spiritual health, and social connection, may pose greater sustainability in the course of this global shift. </div></div>

[STEPS]
?. Survey administered online: "Psychological Well-Being during COVID-19"
?. Data collected in SPSS format for the survey on "Psychological Well-Being during COVID-19" (N = 938 U.S. participants)
TITLE: Efficacy of Simulation-Based Education for intravascular catheterization :protocol for a systematic review and meta- analysis
AUTHORS: Hiromu Okano, Takuya Mayumi, Yuki Kataoka, Masahiro Banno, Yasushi Tsujimoto, Akihiro Shiroshita, Shunsuke Taito, Joho Tokumine

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Background</span></div><div class = "text-block"><span style = "font-weight:bold;">Intravascular catheterization procedures can cause lethal complication.</span></div><div class = "text-block"><span style = "font-weight:bold;"> An appropriate education should be needed for taking advantage of ultrasound guidance, which can realize successful catheterization and prevent mechanical complication. However, the previous review only analyzed central venous catheters, and multiple  randomized controlled trials (RCTs) have been published since then. We will therefore conduct an update review of simulation education for all types of catheters.</span></div><div class = "text-block"><span style = "font-weight:bold;">Aim</span></div><div class = "text-block"><span style = "font-weight:bold;">The purpose of this systematic review is to assess whether simulation education for ultrasound-guided intravascular catheterization will improve the success rate of  catheter insertion compared with no simulation education</span></div><div class = "text-block"><span style = "font-weight:bold;">Methods and analysis</span></div><div class = "text-block"><span style = "font-weight:bold;">We will conduct a systematic review of the relevant literature according to the Cochrane Handbook and the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines. We will include RCTs assessing the effect of simulation education for ultrasound-guided intravascular catheterization.</span></div><div class = "text-block"><span style = "font-weight:bold;">The primary outcomes are success rate and adverse events, and secondary outcomes are first-time puncture success rate in eligible patients and number of punctures to success. Two independent reviewers will extract the data and assess the risk of bias.</span></div><div class = "text-block"><span style = "font-weight:bold;">Ethics and dissemination</span></div><div class = "text-block"><span style = "font-weight:bold;">In this systematic review, only data from previously conducted studies will be analyzed. We will publish this systematic  review in peer-reviewed journals.</span></div></div>

[STEPS]
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol includes a computational pipeline used in assembly and annotation of Kiwifruit Actinidia eriantha genome.

[STEPS]
SECTION: Illumina raw reads cleaning
?. 
SECTION: PacBio assembly
?. 
SECTION: Polish of PacBio assembly with Illumina reads
?. 
SECTION: Scaffold anchoring with Hi-C data
?. 
SECTION: Repeat annotation
?. 
SECTION: Gene prediction
?. 
SECTION: BUSCO evalution
?. 
SECTION: K-mer analysis
?. 
SECTION: Molecular dating and gene family evolution
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Explore and compare two strains of <em>Dehalococcoides mccartyi</em> to discover putative gene(s) responsible for complete dechlorination of PCE to nontoxic end product, ethylene.</p>
<p> </p>

[BEFORE_START]
<p>If you do not have an existing JGI SSO account, make your request here: <a href="https://contacts.jgi.doe.gov/registration/new" target="_blank">https://contacts.jgi.doe.gov/registration/new</a></p>

[GUIDELINES]
<p><strong>Background:</strong></p>
<p> </p>
<p>Organohalides (e.g., tetrachloroethylene, polychlorinated biphenyls, dioxins) constitute a large proportion of environmental pollutants, and reductive dehalogenases encoded by obligate organohalide-respiring bacteria (OHRB), catalyze their dehalogenation to non-toxic metabolites. Corrinoid cobalamin (B12) is an essential cofactor of reductive dehalogenases. De novo biosynthesis of corrinoid is one of the most complicated metabolic pathways in nature, and OHRB have developed different strategies to cope with their need for this cofactor.</p>
<p> </p>
<p><strong>Case Study:</strong></p>
<p> </p>
<p>Explore and compare two strains of <em>Dehalococcoides mccartyi</em> to discover putative gene(s) responsible for complete dechlorination of PCE to nontoxic end product, ethylene.</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" style="display: block; margin-left: auto; margin-right: auto;" src="https://s3.amazonaws.com/pr-journal/ec7c2w6.png" data-src="https://s3.amazonaws.com/pr-journal/ec7c2w6.png" data-ofn="Screen Shot 2016-07-18 at 10.29.21 AM.png" /></p>
<p> </p>
<p><strong>REFERENCES</strong></p>
<ol>
<li>Genome sequence of the PCE-dechlorinating bacterium Dehalococcoides ethenogenes. Seshadri R, et al. Science, 2005 Jan 7. PMID 15637277</li>
<li>Genome sequence of the chlorinated compound-respiring bacterium Dehalococcoides species strain CBDB1. Kube M et. Al. Nat Biotechnol. 2005 Oct;23(10):1269-73.</li>
<li>Microbial species delineation using whole genome sequences, Varghese, N. et. al. Nucl. Acids Res. (2015)</li>
<li>Trichloroethene reductive dehalogenase from Dehalococcoides ethenogenes: sequence of tceA and substrate range characterization. Magnuson JK, et al. Appl Environ Microbiol, 2000 Dec. PMID 11097881</li>
<li>The Genomes OnLine Database (GOLD) v.5: a metadata management system based on a four level (meta)genome project classification. Reddy TBK, et. al., Nucl. Acids Res. (2014)</li>
<li>Discovery of Novel Plant Interaction Determinants from the Genomes of 163 Root Nodule Bacteria. Seshadri, R. et.al. Nat. Sci. Rep. 2015. 5:16825.</li>
</ol>
<p> </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
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?. 
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?. 
?. 
?.
TITLE: Test protocol II
AUTHORS: Abby   Moore

[DESCRIPTION]
This is a test protocol

[STEPS]
1. Use 80:20 MeOH:H2O for this step. This is not easy to access by machine.
2. If you haven't already, make a solution with the following components:

80 % volume   
20 % volume   

3. If you haven't already, make a solution with the following components:

80 % volume   
20 % volume   

4. Use this piece of equipment:

 
 



5. 100 µL   







5.1. To the sample on ice , add
Use   
Use 2 % (v/v)   and 1 % (v/v) sample
TITLE: RNA-seq of primary human fibroblasts cultured on soft and stiff ECM
AUTHORS: Brian D. Cosgrove, Lexi Bounds, Carson Key Taylor, Alan L. Su, Anthony J. Rizzo, Alejandro Barrera, Andrea R Daniel, Gregory E. Crawford, Brenton D. Hoffman, Charles A. Gersbach

[DESCRIPTION]
This protocols describes methods for RNA-seq to characterize the cellular gene expression responses to changes in extracellular matrix stiffness cues. 






[STEPS]
SECTION: Cell culture and soft hydrogel processing
1. Culture primary human neonatal fibroblasts (HFF cells, ATCC CRL-2097) in DMEM with 10% FBS, 1% AntiAnti, and 1% NEAA (Sigma) on tissue culture plastic (TCP).
SECTION: Cell culture and soft hydrogel processing
2. Use polyacrylamide hydrogel 35 and 150 mm PetriSoft EasyCoat dishes (Matrigen) with an Elastic Modulus of 1 kPa (“soft”) and 50 kPa (“stiff”). 
SECTION: Cell culture and soft hydrogel processing
3. Incubate dishes for 5 minutes with sterile PBS, rinse two more times with sterile PBS, followed by the addition of 10 ug/mL fibronectin (Sigma) for 30 minutes at room temperature.
SECTION: Cell culture and soft hydrogel processing
4. Remove fibronectin and rinse dishes twice with sterile PBS, followed by a 20 minutes incubation with complete growth media.
SECTION: Cell culture and soft hydrogel processing
5. Remove media from the dishes and add cell suspensions. Allow cells to attach overnight.
SECTION: Bulk RNA-Seq
6. Seed 40,000 HFF cells on 50 kPa dishes and TCP dishes, seed 70,000 HFF cells on 1 kPa dishes to achieve the same effective plating density due to slightly reduced HFF attachment rates (and spreading) on 1kPa hydrogels.
SECTION: Bulk RNA-Seq
7. 20 hours after seeding, trypsinize cells and spun down at 300g for 5 minutes. Isolate RNA from the cells using the Norgen Total RNA Purification Kit (#17250) according to the manufacturer's protocol.
SECTION: Bulk RNA-Seq
8. Run RNA samples on a RNA TapeStation (Agilent) to verify all samples had a RIN score > 8.
SECTION: Bulk RNA-Seq
9. Build cDNA libraries from RNA inputs using the TruSeq Stranded Library Prep Kit (Illumina #RS-122-2101) according to manufacturer’s instructions. 
SECTION: Bulk RNA-Seq
10. Perform quality control by running the amplified libraries out on a High Sensitivity D1000 Tapestation (Agilent) to confirm expected size. 
SECTION: Bulk RNA-Seq
12. Dilute libraries to 10nM and pool them together in equal volumes. Perform sequencing on an Illumina HiSeq 2500 using a 50bp PE RapidRun kit. 
SECTION: Bulk RNA-Seq
13. Subject resulting reads to adapter trimming using Trimmomatic v0.32 (1), align them to GRCh38 with the STAR v2.4 aligner (2), and retrieve counts using featureCounts (3) from subread version 1.4.6p4 with Gencode v22 gene annotations used as reference.
SECTION: Bulk RNA-Seq
14. Perform differential expression analysis using edgeR quasi-likelihood methodology (4) and visualize data using Degust (5) and Rstudio. Determine genes with significant differential expression using a threshold of FDR < 0.05 and absolute value of Log2(FC) > 1. 
SECTION: Bulk RNA-Seq
11. Perform Qubit dsDNA HS (High Sensitivity) assays to determine a final concentration.
TITLE: U54 SCENT Normal Lung Donor Tissue Collection Procedure
AUTHORS: Matthew Hartwig, Sarah Casalinova*

[DESCRIPTION]
This document outlines the acceptance criteria and workflow for notification, screening, acceptance and logistics of donor lung collection between Duke University Medical Center and our local Organ Procurement Organizations. Donor Lungs accepted for the SCENT protocol arrive to our Surgery Substrate Services Core packaged on ice, en bloc and are processed and banked by a Duke Histologist. 

[STEPS]
SECTION: Inclusion Criteria - Adults and Pediatrics
1. Inclusion Criteria:
    • All ages (if donor is ≤ 18yo refer to peds exclusion list)
    • Brain Dead (DBD) or Death by Circulatory Death (DCD)
    • Is the donor research consented?

Exclusion Criteria - Adults:
    • PCR COVID+ Donors (must have at least 1 follow up negative results for COVID+ within the last                 
       month.  
    • HIV Positive  
    • NAT + HCV-NAT negative is ok  
    • NAT + BV-NAT negative is ok  
    • Emphysema  
    • COPD 
    • Active Lung Cancer  
    • Untreated MRSA  
    • Sepsis     
    • Cause of Death= smoke inhalation
    • Active Asthma
    • Influenza positive
    • Smoking History > 20 packs per year (Document search for “packs” can be helpful to expedite 
        getting data)
    • Aspergillus infection
    • Active tuberculosis
    • Pneumonia (exception-if pneumonia is localized to 1 lobe or 1 side of the lungs, ok to still collect 
       per Dr. Hartwig 12/12/2022, reverified 1/19/2023)

Exclusion Criteria - Pediatrics (donors ≤ 18yo)
    • PCR COVID + Donors (must have at least 1 follow up negative results for COVID + within the last 
       month) 
    • HIV Positive 
    • NAT + HCV-NAT negative is ok  
    • NAT + BV-NAT negative is ok  

NOTE:
*if a donor meets any of the exclusion criteria, turn down acceptance of the lungs for research and leave a comment below explaining which exclusion was met and why*
SECTION: Protocol
2.
TITLE: Lipid (Oil Red O) Staining 
AUTHORS: Laura.SabioRodriguez

[DESCRIPTION]
Lipid droplets (LDs) are dynamic, ubiquitously present lipid-storage  organelles, predominantly present in the adipocytes. Triglycerides,  neutral lipids, and cholesterol esters stored in LDs are the largest  sources of energy. The presence of excess LDs in adipocytes results in  obesity and obesity-linked pathologies such as dyslipidemia and diabetes  type.

[BEFORE_START]
Kit: Lipid (Oil Red O) Staining Kit. SIGMA-ALDRICH, Catalog Number: MAK194
Briefly centrifuge vials before opening. Use ultrapure water for the preparation of reagents.

Reconstitute the Oil Red O Stock solution with 20 mL of  . Mix well and leave undisturbed for 20 min to make the Stock Solution, which is stable for 1 year.

The Oil Red O Working Solution is obtained by adding 3 parts of Oil Red O Stock Solution to 2 parts of water. Mix well and leave undisturbed for 10 min and filter through Whatman No. 1 filter paper. The Working Solution is stable for 2h and must be prepared 15 min before use.

[STEPS]
SECTION: Procedure
2. Fixation
Remove the medium and gently wash the cells twice with  . Add   to the cells and incubate for 30 min.
Note: Do not add formalin directly onto the cells. Pipette onto the wall and mix gently rotating.

SECTION: Procedure
1. All components:
Use 100 µL for a 96 well plate
Use 2 mL for a 6 well plate
Use 6 mL for a 100 mm culture dish

SECTION: Procedure
3. Discard the formalin and wash the cells twice using  . Add 60%   to the cells and incubate for 5 min. 

SECTION: Procedure
4. Discard 60% isopropanol and cover the cells evenly with Oil Red O Working Solution. Rotate the plate or dish, and incubate for 10 min. 

SECTION: Procedure
5. Discard the Oil Red O Solution and wash the cells 2-5 times with   until no excess stain is seen.

SECTION: Procedure
6. Add   to the cells and incubate for 1 min. Discard hematoxylin and wash the cells 2-5 times with  .

SECTION: Procedure
7. Cover the cells with   and view under microscope. Lipid droplets appear red and nuclei appear blue.
Note: Keep the cells covered with water to avoid drying.
TITLE: in situ hybridization
AUTHORS: 张 雪

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

[STEPS]
?. [Day1]
复水：75%MetOH+25%PBT 1ml shake for 5min
?. [Day1]
50%MetOH+50%PBT 5min
?. [Day1]
25%MetOH+75%PBT 5min
?. [Day1]
100%PBT 5min 1/4
融化PK
?. [Day1]
100%PBT 5min 2/4
?. [Day1]
100%PBT 5min 3/4
?. [Day1]
100%PBT 5min 4/4
?. [Day1]
透化：Proteinase K: PBT = 1 : 1000 (终浓度10µg/ml），24h前：不消化；24h：5min；36h：10min；48h：15min；60h：20min；72h及以上：30min
?. [Day1]
再固定：4%PFA 30min
加PFA时，加完一管应立即放在摇床上，否则容易在底部凝结成团
?. [Day1]
1×PBT 5min 1/4
?. [Day1]
1×PBT 5min 2/4
?. [Day1]
1×PBT 5min 3/4
?. [Day1]
1×PBT 5min 4/4
?. [Day1]
预杂交：500µl Hybe buffer（需提前预热），68.5°C水浴锅中预热2−5h
68.5 °C
?. [Day1]
杂交：6µl probe+200µl Hybe buffer，68.5°C overnight
68.5 °C
?. [Day2]
回收探针，保存在-20°C，可重复使用（以下步骤仍在水浴锅中进行，且不用盖盖子，根据热胀冷缩原理，所吸液体量减至700-800µl）
750 µl
68.5 °C
提前配制以下液体，并放在68.5°C水浴锅中预热，29）之前的操作均在水浴锅中进行
?. [Day2]
Hybe buffer 10min
?. [Day2]
75% Hybe buffer+25% 2×SSCT 10min
?. [Day2]
50%Hybe buffer+50% 2×SSCT 10min
?. [Day2]
25%Hybe buffer+75% 2×SSCT 10min
?. [Day2]
2×SSCT 10min
?. [Day2]
0.2×SSCT 15min 1/4
?. [Day2]
0.2×SSCT 15min 2/4
?. [Day2]
0.2×SSCT 15min 3/4
?. [Day2]
0.2×SSCT 15min 4/4
以下步骤在室温水平摇床上进行
?. [Day2]
75% 0.2× SSCT+25% MABt 5min
0 Room temperature
1 mL
?. [Day2]
50% 0.2× SSCT+50%MABt 5min
?. [Day2]
25%0.2× SSCT+75%MABt 5min
?. [Day2]
MABt 5min
?. [Day2]
1×blocking 500µl（每管配700µl）2h，RT
0 Room temperature
500 µl
?. [Day2]
Anti-Dig-AP 1:2000，200µl，4°C摇床缓慢摇动过夜
4 °C
200 µl
注意与FISH不同，此处为Anti-Dig-AP
?. [Day3]
回收抗体，保存在4°C冰箱
?. [Day3]
1×MABt 15min 1/8
?. [Day3]
1×MABt 15min 2/8
?. [Day3]
1×MABt 15min 3/8
?. [Day3]
1×MABt 15min 4/8
?. [Day3]
1×MABt 15min 5/8
?. [Day3]
1×MABt 15min 6/8
?. [Day3]
1×MABt 15min 7/8
?. [Day3]
1×MABt 15min 8/8（配制NTMT，现配现用，每管需要3.5ml）  AB120mlNTMT22ml1M pH9.5 Tris-HCl32ml1M NaCl40.5ml10% Tween515.5mlddH2O
AB120mlNTMT22ml1M pH9.5 Tris-HCl32ml1M NaCl40.5ml10% Tween515.5mlddH2O
?. [Day3]
Equilibrate with NTMT，5min 1/3
?. [Day3]
转移至24孔板，NTMT 5min 2/3
配显色液：BCIP in NTMT（1:50，500µl每管）
?. [Day3]
NTMT 5min 3/3
?. [Day3]
显色：BCIP in NTMT（1:50，500µl），37°C避光，每隔15min观察一次（BF，磨砂玻璃）
500 µl
37 °C
避光
?. [Day3]
1ml stop solution 终止反应后，将胚胎置于甘油中拍照
TITLE: Interviewing for Art as an Act of Resistance 
AUTHORS: angelabelulia

[DESCRIPTION]
To gain insight and explore how art has served as an act of resistance against French Colonization in French Polynesia

[STEPS]
1. Understand the purpose of the interview: To gain insight and personal experiences into how music has served as an act of resistance against French Colonization in French Polynesia
2. Identify the stakeholders: local artists, cultural leaders/elders and community organizations 
3. Ask for permission and consent to research within the community
4. Invite to participate in interviews
5. Schedule interviews: one interviewees have agreed to participate, schedule a time and place for interview. 
6. Prepare a set of questions that aim to gather perspectives and personal experiences/stories about how language and music has had and impact/influence on their identity and sense of self/belonging
7. When the interview day comes, conduct the interview. Ensure to begin. the interview by introducing yourself and explaining the purpose of the interview and ask for consent to record the conversation.
8. Once the interview is complete, thank the interviewee for their time and contribution.
9. After the interview process is done, place all the audios into otter.ai to transcribe and MAXQDA to sift through key points. 
10. With otter.ai, go through each audio recording and read through the transcript. Fix any errors that the software might have made to ensure that the data will be accurate and clean.
11. In MAXQDA, develop a set of labels that one can code the interviews to find similarities and differences within each personal experience. 
12. Report findings in a clean and clear manner. Make sure to cite sources and relevant context.