eamag/protocols-with-steps huggingface dataset:
TITLE: Cell harvest and RNA prep using Qiagen RNAeasy Kit
AUTHORS: Katrin Blondrath

[STEPS]
?. [Cell harvesting and RNA extraction ]
Aspirate medium from 6 well plates, wash once with PBS
?. [Cell harvesting and RNA extraction ]
Add  lysis buffer containig freshly added beta-mercaptoethanol to each well (lysis buffer provided in RNAeasy kit)
500 µl
Beta-mercaptoethanol should be added freshly every time performing the experiment
?. [Cell harvesting and RNA extraction ]
(OPTIONAL) Wrap plates in parafilm, store at until extraction
-80 °C
?. [Cell harvesting and RNA extraction ]
(Defrost plates and) Scrap with 1000 ul pipette tip and pipett up and down the well, transfer into low bind nuclease-free tube
?. [Cell harvesting and RNA extraction ]
Extract according to manufacturer's protocol using Qiagen RNAeasy kit (including DNAse treatment step)
?. [Storage]
Measure RNA concentration using Nanodrop and store at or continue with qPCR
-80 °C
?. [qPCR]
Dilute samples to have equal concentrations of 2500 ng of each sample. Use   of this diluted RNA directly in 10 ul reaction to achieve 50nM per well of 384 well reaction plate
2 µl
?. [qPCR]
Mix contents of One Step TB Green™ PrimeScript™ RT-PCR Kit II (in case running a whole 384well plate)Pre Master: Mix contents of entire kit: TB Green buffer RT/Polymerage enzyme ROX  H2OAdjust accordingly to the total number of required reactions1x Mastermix:  ABC1&nbsp;&nbsp;1x in micro-liters2ENZYME&nbsp;0.43TB green Buffer 4&nbsp;&nbsp;54Primer 30uM F/R mix&nbsp;0.85RNA&nbsp;26ROX&nbsp;0.47H2O&nbsp;1.8
2500 µl
200 µl
100 µl
800 µl
[total]
ABC1&nbsp;&nbsp;1x in micro-liters2ENZYME&nbsp;0.43TB green Buffer 4&nbsp;&nbsp;54Primer 30uM F/R mix&nbsp;0.85RNA&nbsp;26ROX&nbsp;0.47H2O&nbsp;1.8
?. [qPCR]
Final master mix:Per reaction/ per well of 384well plate = Multiply by amount of samples and add ~20% dead volume.
10 µl
TITLE: Mammalian Cell Culture: Subculturing
AUTHORS: Kenneth Schackart

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details how to subculture/passage nearly confluent mammalian cells grown in a tissue culture flask.</div></div>

[STEPS]
?. [Assess Cell Confluency]
Under light microscope, look at the cells and assess level of confluency. This is how you will determine the need to subculture.
Confluency can be estimated by evaluating the percentage of surface covered by cells.
?. [Wash Cells]
Using serological pipette, add  DPBS to flask. [ for T-25]
4 ml
1 ml
?. [Wash Cells]
Using serological pipette, remove DPBS and dispose into waste beaker.
?. [Wash Cells]
Repeat the above 2 steps, so that you will wash the cells twice.
Always use a fresh pipette tip when drawing liquid from a stock solution.
?. [Trypsinize]
Add warmed trypsin-EDTA to T-25 flask. [ for T-25]
4 ml
1 ml
?. [Trypsinize]
Wait approximately for trypsin-EDTA to detach the cells.
This time will vary in practice, and depends on cell type and trypsin concentration (i.e. 0.05% vs 0.25%). Some cell types may take up to 15 minutes. In those cases, assess detachment progress using a light microscope.
For cell types that take longer to detach, place flask in incubator to keep the temperature high enough for trypsin to remain active.
?. [Trypsinize]
Add cell culture media. [ for T-25]
4 ml
1 ml
Trypsin-EDTA is neutralized by adding a volume of cell culture media equal to that of trypsin-EDTA.
?. [Spin Down]
Using a serological pipette, transfer the cell suspension (cells, trypsin-EDTA, and cell culture media) into a 15 mL centrifuge tube.
?. [Spin Down]
Centrifuge the cell suspension on  for .
?. [Resuspend and Reseed]
Remove supernatant, dispose into waste beaker.
You can leave a small amount with the serological pipette, the rest will be taken off in the next step.
?. [Resuspend and Reseed]
Using a 1000 μL pipette, carefully remove the remaining supernatant, being cautious not to disturb the cell pellet.
Always dispose of pipette tips in sharps container. Do not use the same tip twice.
?. [Resuspend and Reseed]
Add cell culture media to the cell pellet, and allow to sit for .
1000 µl
?. [Resuspend and Reseed]
Gently pipette mix the cell pellet until the pellet is resuspended.
Pipette mixing is done by slowly drawing in solution and pushing it out several times, all without removing the pipette tip from the solution. Ask T.A. for help on this if you need some pointers.
?. [Spin Down]
Add fresh cell culture media to T-25 flask, this will preserve any remaining cells.
9 ml
?. [Resuspend and Reseed]
Seed 2 flasks each with cell suspension.
500 µl
You may seed more than 2 flasks, just use smaller volumes in each.
?. [Incubate]
Incubate at in CO2 incubator.
?. [Wash Cells]
Remove media from flask.
?. [Resuspend and Reseed]
Label flask with updated passage number along with the date.
TITLE: Lysosomal membrane integrity
AUTHORS: Veerle Baekelandt

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Lysosomal membrane integrity </span></div></div>

[STEPS]
?. seed cells (SHSY-5Y) for 24:00:00 in a 12 well plate  at a density of 1 x 105 cells per well
?. incubate cells with 5 µg/ml acridine orange (disolved in media) for  at
37 °C
?. discard medium and wash cells with  of 1X PBS. Add fresh medium to the wells.
1 mL
?. treat cells with SPM (different concentrations: 0.001, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5 and 10 mM)) and positive control with chloroquine (500 µm) for at
37 °C
?. collect cells and resuspend in 1X PBS + 1% BSA.
?. capture the mean fluoresence of 10 000 events with a flow cytometer.
TITLE: Direct cDNA synthesis and pre-amplification of single embryos for RT-PCR
AUTHORS: Yao Xiao, Peter Hansen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol allows gene expression analysis within single embryos on multiple targets in either conventional real-time PCR or using the Fluidigm platform. The protocol makes extensive use of the CellsDirect Kit from ThermoFisher Scientific (catalog number 11753). References to the kit refer to this product. This protocol describes a method that converts RNA directly into cDNA without RNA extraction, which avoids the variation due to extraction process. The number of genes of interest can be up to more than 100. However, the target genes for analysis have to be determined prior to cDNA synthesis. Once the cDNA is made, there is no way to add additional gene to analyze. The best approach to primer design is to have primers span an intron in the target region. This is not a requirement, however, because of the DNase treatment included in the protocol.</div></div>

[STEPS]
?.
TITLE: GeoMx Digital Spatial Profiler (DSP) Protocol v2 - University of Minnesota TMCs
AUTHORS: Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
The nanoString GeoMx‱ Digital Spatial Profiler (DSP) is a platform that allows high-plex profiling at the protein and RNA level, providing spatial and temporal assessment of tumors in frozen or formalin-fixed paraffin-embedded limited tissue sample.

The following protocols/manuals were used at the University of Minnesota TMCs in collaboration with the University of Minnesota Genomics Center. 

[STEPS]
SECTION: Slide Preparation
1. GeoMx DSP Manual Slide Preparation User Manual
   

SECTION: ROI Acquisition
2. GeoMx DSP Instrument User Manual
  

SECTION: Library Preparation
3. GeoMx DSP NGS Readout User Manual
  
 



SECTION: FASTQ Generation
4. BCL data from Illumina sequencer is demultiplexed and converted into FASTQ format using bcl2fastq version 2.20.0
TITLE: Analyze Repeats
AUTHORS: Karina Jhingan

[DESCRIPTION]
A protocol that uses Homer's analyzeRepeats, findGo, and FindMotifsGenome on the output of bowtie2 run on data using different pre-treatment conditions (LSD1i, TGFb, and LDS1i+TGFb) at 3 different timepoints (day 0, 8, 16).

[STEPS]
SECTION: Introduction
1. This linux analysis is using Homer, linked below. Here we are only focusing on the H3K9me3 histone for the human genome to compare different pre treatment conditions at different time points.   http://homer.ucsd.edu/homer/ngs/analyzeRNA.html
SECTION: Load modules
2. I have found that the order that modules is loaded in can cause some errors later when using certain Homer functions and found that this order doesn't cause any errors. if your analysis is on an organism other than one of the preloaded organisms in Homer and you don't have write access, you likely will have to download Homer (the steps for doing this can be found in my CUT&tag protocol).

You can play around with loading or not loading samtools a second time as in line 4 of the below code, I found that this helped with certain Homer functions.
 

SECTION: Set variables
3. 
 

SECTION: Run AnalyzeRepeats
5. Here we first make a tag directory using Homer's function for each sample where the input is a mapped bam data file (from Bowtie2).
Because we are interested in entire gene bodies, the -count parameter would be "genes".

We will output the results into $projPath/analyzeRepeats/${org}_${sample}.txt

We will be running analyzeRepeats 3 times for each sample as we want to compare all time points (D0vD8, D8vD16, D0vD16
 

SECTION: Set variables
4. Download genome reference file
 

SECTION: findGo (on top 1k differentially expressed genes)
6. 
Documentation: http://homer.ucsd.edu/homer/microarray/go.html

Here we are preparing the files to use as an input for findGo (gene ontology analysis) by finding the log fold change on our outputs from the previous step.

Here we calculate log fold change (lfc), sort the resulting file by lfc. Then we select relevant columns and take only the top 1000 rows. 

We perform this 3 times per loop as we have the three different time comparisons as mentioned in the previous step, after that we run homer's findGo on these selected differentially expressed genes. 

 

SECTION: Find Motifs
7. Documentation: http://homer.ucsd.edu/homer/ngs/peakMotifs.html

Also using the output of analyzeRepeats, we will select the columns in the correct orde in order to turn the text file into a bed file to input into findMotifsGenome from Homer. 

Parameters: We just used the basic parameter for region size, although it is helpful to know the regions size specific to your experiment and use that instead. -p 4 refers to the number of cores to use, this function take a while to process, and I found that 4 was the optimal number of cores in our case, feel free to experiment going up and down in number of cores to speed up the time. 
 

8.
TITLE: Cell surface biotinylation
AUTHORS: rosanne.wouters, Peter Vangheluwe

[DESCRIPTION]
The protocol describes cell surface biotinylation to identify plasma membrane localized proteins in cell culture via western blotting.

[STEPS]
1. grown cells until 70-80% confluency in 10cm dish 
3. incubate cells for 30 min c on ice with PBS containing 2.5 mg/ml Sulfo-NHS-SS-biotin (Pierce).
4. stop the biotinylation reaction by washing 3 times for 5min with quenching solution (0.5% BSA and 100 mM glycine in PBS).
5. collect cells by scraping in PBS and centrifugation 500 rpm, 5 min, 4 °C 

7. clear cell lysate by centrifugation (20min, 14000 gavg, 4°C) and collect supernatant
8. isolate biotinylated proteins   with immobilized Neutravidin beads
9. Input and bound proteins were processed for Western blotting with 4x LDS loading buffer.
2. place cells on ice and wash with ice-cold PBS
6. lyse cells by resuspending cell pellet RIPA buffer supplemented with protease inhibitor and incubating for 30min on ice
8.1. wash neutravidin beads 2 times with RIPA buffer
8.2. add supernatant from step 7 to beads
8.3. incubate while rotating head-over-head 120 min

8.4. wash neutravidin beads 3 times with RIPA buffer
9.1. remove all RIPA buffer from neutravidin beads
9.2. add 4x LDS loading buffer 
9.3. denature proteins for 10min, 70°C
10. proceed with Western blotting
TITLE: Optimum Keto Pills Reviews [Official-2022] Ratings &amp; Results!
AUTHORS: Optimum Keto

[DESCRIPTION]
Optimum Keto

[STEPS]
1. Optimum Keto Pills
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2.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Successful transformation of <em>Phaeodactylum tricornutum </em>(CMP632) by electroporation was achieved based on a modified version of Zhang &amp; Hu (2014). </p>

[BEFORE_START]
<p>Materials needed for transformation                                                  </p>
<ul>
<li>375 mM Sorbitol, filter sterilize and store at -20°C.</li>
<li>Plasmid DNA --&gt; linearize with ScaI at 37°C (5’…AGT|ACT… 3’) (3’…TCA|TGA…5’)</li>
<li>Salmon sperm dna--&gt;  denature the DNA by boiling for 1 minute, then place on ice</li>
<li>f/2 medium + Si</li>
<li>solid medium (50% ASW containing f/2 nutrients, 1.2% agar + zeocin 75 µg/ml)</li>
</ul>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Q5® Mastermix PCR-OpenPlast
AUTHORS: yasoo 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is for PCR with Q5® High-Fidelity 2X Master Mix (M0492)</div></div>

[STEPS]
?. Set up the following reaction on ice:  ABCD12Q5 High-Fidelity 2X Master Mix5 µl10 µl1X310 µM Forward Primer0.5 µl1 µl0.5 µM410 µM Reverse Primer0.5 µl1 µl0.5 µM5Template DNAvariablevariable6Nuclease-Free Waterto 10 µlto 20 µl
ABCD12Q5 High-Fidelity 2X Master Mix5 µl10 µl1X310 µM Forward Primer0.5 µl1 µl0.5 µM410 µM Reverse Primer0.5 µl1 µl0.5 µM5Template DNAvariablevariable6Nuclease-Free Waterto 10 µlto 20 µl
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?. Gently mix the reaction.
?. Collect all liquid to the bottom of the tube by a quick spin if necessary and overlay the sample with mineral oil if using a PCR machine without a heated lid.
?. Quickly transfer PCR tubes to a thermocycler preheated to the denaturation temperature (98°C).
TITLE: cDNA synthesis  
AUTHORS: miquel.vila

[DESCRIPTION]
High-Capacity cDNA Reverse Transcription Kits(#4368814, Applied Biosystems - ThermoFisher)

[STEPS]
SECTION: cDNA synthesis
1. Prepare the 2X master mix (ul per sample):
- 2ul RT Buffer
- 0.8ul 25X dNTP Mix(100mM)
- 2ul Random primers
- 1ul Multiscribe Reverse Transcriptase
- 4.2ul Nuclease free water
SECTION: cDNA synthesis
3. Mix the 2X master mix (10ul) with the RNA samples (10ul) and mix by pippeting
SECTION: cDNA synthesis
4. Centrifuge the plates to spin down all the reagents.
SECTION: cDNA synthesis
5. Keep the samples on ice
SECTION: cDNA synthesis
6. Program the thermal cycler to 25C for 10min 37C for 120 mintues 85C for 5 minutes and 4C until finished.
SECTION: cDNA synthesis
7. Store the cDNA samples at -20C for short-term storage or -80C for long-term storage (years).
SECTION: cDNA synthesis
2. Take 0.5ug of RNA in a maximum of 10ul of sample volume
TITLE: Fixation and immunocytochemistry (fluorescence) in PFF-treated cultures
AUTHORS: Maria Iuliano

[DESCRIPTION]
This protocol outlines methods used for the preparation of in vitro models for fluorescent imaging including primary rat and mouse hippocampal cultures treated with PFFs.

[BEFORE_START]
Please read safety guidelines ahead of working with PFFs

[GUIDELINES]
Parameters used in this protocol may be optimized and modified for individual use depending on experimental needs.

[STEPS]
SECTION: Fixation and Immunocytochemistry (Fluorescence)
1. In a cell culture hood remove media and wash briefly with 1x PBS.
 

SECTION: Fixation and Immunocytochemistry (Fluorescence)
2.   Fix using 4% PFA 4% sucrose in 1xPBS with gentle rocking/shaking.
 

SECTION: Fixation and Immunocytochemistry (Fluorescence)
3.   Wash 1x PBS. Repeat 3 times.
 

SECTION: Fixation and Immunocytochemistry (Fluorescence)
4.   Block in blocking buffer (5% NGS, 0.3% Triton-X in 1x PBS)
SECTION: Fixation and Immunocytochemistry (Fluorescence)
5.     Incubate in solution containing primary antibodies diluted in 5% NGS in 1x PBS
 

SECTION: Fixation and Immunocytochemistry (Fluorescence)
6.   Wash 1x PBS. Repeat 3 times.
SECTION: Fixation and Immunocytochemistry (Fluorescence)
7.   Incubate in solution containing secondary antibodies diluted in 5% NGS in 1x PBS
 

SECTION: Fixation and Immunocytochemistry (Fluorescence)
8.   Wash 1x PBS. Repeat 3 times.
SECTION: Fixation and Immunocytochemistry (Fluorescence)
9. Mount coverslips onto microscope slides using    or preferred mounting medium.
SECTION: Fixation and Immunocytochemistry (Fluorescence)
10.  Let cure 2880 min before imaging
TITLE: Hemolysis measurement of plasma/serum samples with Nanodrop ND1000
AUTHORS: Hetty Helsmoortel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to measure hemolysis on plasma or serum samples. </div></div>

[STEPS]
?. [Before you start]
Place: room 120.042, 2nd floor MRB1Aliquot 3 ul plasma sample in a separate tube (can be left at room temperature)Open sample arm and clean sample pedestal with an ethanol-dipped swab (dust-free). Swabs are standing next to the machine.Clean the sample pedestal with a dry swab (dust-free).
?. [Preparing hemolysis measurement]
start ND-1000 softwarechoose ‘Cell cultures’ change max. absorbance from 0.2 to 2 (rescales the vertical axis)change wavelength (lambda) to 414 nmchange max. absorbance from 0.2 to 2 (rescales the vertical axis)change wavelength (lambda) to 414 nm
?. [Load blank sample]
pipet 1 ul water to the sample pedestal, using reverse pipetting to avoid bubbles (use an unused water tube)gently close the sample armclick ‘blank’ to calibrate the machine. If the wave shows peaks, repeat the pipetting and re-blank
?. [Measure blank sample]
open sample arm and clean pedestal with a dust-free swab(to have a good reference, the first sample we measure is always a water sample)pipet 1 ul water to the sample pedestal, using reverse pipetting to avoid bubbles (use an unused water tube)gently close the sample armclick ‘measure’ to measure water sample
?. [Measure plasma/serum sample(s)]
open sample arm and clean pedestal with a dust-free swabpipet 1 ul plasma sample to the sample pedestal, using reverse pipetting to avoid bubblesgently close the sample armclick ‘measure’ to measure first sample(repeat steps above for other samples)clean sample pedestal and close sample arm
?. [Export results]
TITLE: Degassing Reagents
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used for the degassing of critical reagents used in both the colorimetric and fluorescent in situ hybridization (ISH). This is crucial in eliminating hybridization bubbles from forming on the tissue.  Hybridization bubbles result in no expression where they are located on the tissue.</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: SapphireAmp PCR Master Mix -- CHEM 584
AUTHORS: Ken Christensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">SapphireAmp Fast PCR Master Mix contains a hot start PCR enzyme, optimized buffer, dNTP mixture, gel loading dye (blue), and a density reagent as a 2X premix. SapphireAmp Fast PCR Master Mix is optimized for fast PCR and offers a rapid extension rate (10 sec. per kb). The inclusion of blue dye and a density reagent allows direct loading of PCR products on an agarose gel for electrophoresis. The master mix format simplifies workflows and sample handling; simply add primers, template, and water and then begin PCR. SapphireAmp Fast PCR Master Mix is ideal for fast colony PCR screening. Fast colony PCR amplification of a 5 kb insert can be completed in approximately 1 hr 15 min. Furthermore, it is possible to amplify fragments up to 6 kb from genomic DNA templates.</div></div>

[STEPS]
?. [Setup Reaction]
To a  aliquot of SapphireAmp PCR Master Mix, add template (4 µl cleared lysate for colony PCR or  of purified DNA for typical PCR), forward and reverse primers to a final concentration of . Adjust final volume to  with nuclease free water or autoclaved water.  AB1Sapphire Master Mix12.5 ul (pre-aliquoted and stored in the freezer)2Template4 ul of bacterial lysate or 3Forward Primer0.5 ul of 10 uM primer dilution4Forward Primer0.5 ul of 10 uM primer dilution5ddH2Oto a final volume of 25 ul
12.5 µl
1 ng
25 µl
AB1Sapphire Master Mix12.5 ul (pre-aliquoted and stored in the freezer)2Template4 ul of bacterial lysate or 3Forward Primer0.5 ul of 10 uM primer dilution4Forward Primer0.5 ul of 10 uM primer dilution5ddH2Oto a final volume of 25 ul
?. [Run Reaction]
followed by 30 cycles of 98°C, 5 sec; 55°C, 5 sec; and 72°C, 40 sec.  ABC1Initial denature98C1 minute2Denature94C10 seconds3Anneal55C10 seconds4Extension72C30 seconds5Repeat steps 2-430-40x6Final extension721 minute7Cool4CUntil cancelled
ABC1Initial denature98C1 minute2Denature94C10 seconds3Anneal55C10 seconds4Extension72C30 seconds5Repeat steps 2-430-40x6Final extension721 minute7Cool4CUntil cancelled
TITLE: iPSC Differentiation
AUTHORS: Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol explains the iPSC differentiation of lines ND1014, N1, and ND27760 from Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons. </div></div>

[STEPS]
?. [Day 1-3]
Plate iPSCs at a density of 1.5 × 105cells per cm2 in a 12‐well plate pre-coated with growth-factor-reduced Matrigel.
Culture medium should be changed daily, and gradually shifted from mTeSR1 to N2 (Thermo Fisher Scientific) in 3 days. The medium also contains  ,  , and  .
?. [Day 1-3]
Transfect iPSCs with A-SA mRNA for 3 days, changing media daily.
?. [Day 4]
Transfect iPSCs with N-SA mRNA for 1 day.
?. [Day 5]
Dissociate cells using Accutase.
?. [Day 5]
Cells can also be cryopreserved in medium containing 40% neurobasal medium with B27 supplement, 50% fetal bovine serum and 10% DMSO.
?. [Neuron Culture]
Change media after 48 hours to remove unattached cells followed by half change every 3–4 days during in vitro maturation.
?. [Neuron Culture]
Detect cell proliferation and death analysis using MTT and LDH kits, respectively.
?. [Day 5]
Replate with neuron medium in poly‐d‐lysine/laminin‐coated plates at the density of 1 × 105cells per cm2.
Neuron medium contains: neurobasal medium with B27 supplement,  ,  ,  ,  ,  , and  .