eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Filtering protocol adapted from SangHoon Lee

[GUIDELINES]
Filtering protocol adapted from SangHoon Lee<strong><br /><br />Materials needed for cruise:</strong> <br />4 acid washed 20L cubitainers <br />8 60cm pieces of acid washed tubing <br />Gloves <br />Sharpie <br />2 coolers large enough to hold all 4 cubitainers<br /><br /><a href="https://s3.amazonaws.com/pr-journal/uwc2xe.png" target="_blank"><img src="https://s3.amazonaws.com/pr-journal/uxc2xe.png" alt="" /><br /><br /><br /></a>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>connect to localhost ip: 127.0.0.1</p>
<p>get deafult ip getway</p>
<p>Get access to root </p>
<p>get permmision </p>
<p>create dump file </p>
<p>run as administrator</p>
<p>open=start adb server </p>
<p>start=exit user </p>
<p> </p>

[STEPS]
TITLE: Luminex-Based Multiplex Cytokine Analysis
AUTHORS: michela.deleidi, Bianca Marchetti, Federico Bertoli, Carmela Giachino

[DESCRIPTION]
Multiplexed kit "Mouse High Sensitivity T Cell Magnetic Bead Panel" allows a Simultaneous analyze low levels of cytokine and chemokine biomarker with the High Sensitivity Bead-Based Multiplex Assays using the Luminex technology, in mouse serum, plasma and cell culture samples.

[STEPS]
SECTION: Luminex-Based Multiplex Cytokine Analysis
1. Measure Cytokines from murine plasma samples, including IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-17A, and TNF-α simultaneously using the multiplexed kit "Mouse High Sensitivity T Cell Magnetic Bead Panel" (MHSTCMAG-70K, MILLIPLEX, EMD Millipore Corp., Billerica, MA, USA). 
SECTION: Luminex-Based Multiplex Cytokine Analysis
2. Perform the assay according to the manufacturer's instructions with an   incubation step (17 hours) at 4 °C. 

SECTION: Luminex-Based Multiplex Cytokine Analysis
3. Analyze samples as single values and use two controls with corresponding acceptance ranges, provided with the kit, for quality control purposes.
SECTION: Luminex-Based Multiplex Cytokine Analysis
4. Perform the readout on a FLEXMAP 3D® instrument (Luminex Corp., Austin, TX, USA).
6. Use the Bio-Plex ManagerTM software (version 6.2) (Bio-Rad, Hercules, CA, USA) for back-calculation of unknown cytokine concentrations.
SECTION: Luminex-Based Multiplex Cytokine Analysis
5. Acquire data using Luminex xPONENT® software (version 4.3) and determine mean fluorescence intensity (MFI).
TITLE: Nuclease Test (OpenVent polymerase, PCR Master Mix, DNA loading dye)
AUTHORS: Nadine          Mowoh, Stephane  Fadanka

[DESCRIPTION]
Quality control and nuclease test involves incubating the enzymes or reagents in reconstituted recombination assays to eliminate the possibility of relevant protein or nucleic acid contaminants that may interfere with recombination assays and lead to misinterpretation of experimental data. Here batch by batch quality controls are done for each new product before it is released to the market. 

This protocol describes the steps in carrying out Nuclease test for:

OpenVent Enzyme
PCR Master Mixes - All formulations (Wet and Dry)
6x DNA loading dye

[BEFORE_START]
Clean and disinfect all work surfaces with a 1:10 dilution of bleach followed by 70% alcohol.
Make sure all resources needed are prepared.

[GUIDELINES]
Before carrying out quality control on products research should be carried out on the possible assays that apply for that particular product and availability of resources.
This protocol describes how to check Nuclease activity for PCR Enzymes, PCR Master Mix and DNA loading dye.

[STEPS]
SECTION: Nuclease activity
1. 
 

SECTION: Nuclease activity
2. Pipetting

Pipette the following reagents into 0.2 mL PCR reaction tubes as shown in the table below, while working on ice.

 


 

SECTION: Nuclease activity
4. 
Checking and interpreting results:

After the incubation period, prepare a 1.5% agarose gel, run and visualized as described in this protocol.

Supercoiled DNA should run faster (as would be expected in the negative control lane). Meanwhile, linear DNA should run true to size (known size of the plasmid used),  and relaxed circle DNA should run slower in a single strand cut (as would be expected in the positive control lane).

Any change in the band intensity and migration position of the plasmid DNA band in the test sample lane compared to that of the negative control signals potential endonuclease activity of the test sample.
SECTION: Nuclease activity
3. Incubation
Incubate reaction tubes for the specified durations. 
After the incubation period, remove the tubes from the water bath and briefly place them on ice to stop any further the reaction.
TITLE: Nuclei isolation from snap frozen human pancreatic tissue using a citric acid buffer
AUTHORS: Luca Tosti, Christian Conrad

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A protocol to isolate nuclei from snap-frozen pancreatic tissue and protect RNA from RNAse-mediated degradation</div></div>

[STEPS]
?. Incubate the tissue for 5 min on ice
[on ice]
[incubation]
?. Crush the tissue with 5 more strokes of the loose pestle and leave to incubate for 5 minutes
[on ice]
[incubation]
?. Homogenize the tissue with 3 more strokes of the loose pestle and 5 more strokes with the tight pestle
?. Filter through a FACS-tube with cell strainer cap (35 μm)
?. Wash filter with 250 μL of S25 buffer
[S25 buffer]
?. Transfer the nuclei suspension to a 1.5 mL eppendorf tube and centrifuge at 4°C, 500g for 5 minutes
[centrifugation]
You will probably not see the pellet, but nuclei are on the wall of the tube! Be careful when aspirating, leave a few μL of liquid at the bottom in the next step!
?. Carefully remove the supernatant, resuspend in 300 μL S25 buffer and load 10 μL of nuclei suspension in a Countess™ Cell Counting Chamber Slide. Count the Hoechst-positive nuclei. Use the brightfield to evaluate the amount of debris present in the suspension and the overall integrity of the nuclei. For an example of Countess images, please see the expected results in Step 13.
[S25 buffer]
?. Resuspend in appropriate volume of Resuspension Buffer. The volume can be determined based on the desired final concentration; 20% of the nuclei might be lost during the previous centrifugation step.
?. Count nuclei and proceed with FACS sorting of the nuclei or directly to the encapsulation in oil droplets.
?. Centrifuge the nuclei at 4°C, 500g for 5 minutes
[centrifugation]
?. Transfer the tissue in the douncer, crush it with one stroke of the loose pestle and dislodge the tissue from the bottom using a P200 tip if necessary
?. Add 1 mL of cold S25 buffer to the douncer placed on ice
[S25 buffer]
?. 
?. Resuspend the nuclei in 1 mL of S25 buffer and repeat the centrifugation step as in step 8.
[S25 buffer]
[centrifugation]
TITLE: DNA functionalization on oxide
AUTHORS: hnyein 

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

[STEPS]
?. [Glass slide cleaning]
Clean glass slides in acetone, IPA by sonicating (2 times each) and put in water and dry under N2. Acetone, IPA in cabinet under fumehood.After cleaning, put under UV for 60 mins. Check the marked level to be consistent every time.
?. [Silanization]
Put glass slides in a dessicator. Put APTMS (3-Aminopropyl)trimethoxysilane on one glass slide and in dessicator. Leave it for 16 h. Take it out and put it in oven for 1 h at 100 C. After this step, the glass slides can be kept sealed in freezer for a long time if cannot perform next step immediately. APTMS on the shelve (row 5). syringes between row 1 and 2 cabinet. needles row 4 middle part.
?. [DNA attachment]
Prepare controlled and test samples on glass slides. EDC and DNAs in freezer, MES in fridge. EDC is in powder format. Make 100mg/mL of EDC in 0.1M MES. NOTE: once EDC is dissolved in 0.1M MES, do everything very quickly because EDC lifetime is short once dissolves in water.  (100uM) polyT: 4.5 uL polyT + 6.2 uL 0.1M MES + 4.3 uL EDC (300uM) HLA: 1.5 uL HLA + 9.2 uL 0.1 MES + 4.3 uL EDCOn glass slides, put PDMS and wells for flowing above solns onto desired spots.Flow above solns and leave them in ice for 2 h. Obtain ice from across the room.
?. [Passivation]
Remove all the liq inside. Wash with DI for 3-4 times and leave it in DI for 5 mins. Remove all the PDMS. and put new black chamber + PDMS. Make 40% PEG: 100/68*weight of PEG powder= volume(uL) of DI need to be added. 11 ul NaHCO3 + 89 uL 40 % PEG Flow the solution into the chamber and leave it for another 2 h in dark, RT. NaHCO3 (use stock conc. 0.89M) on shelves where chlorides solutions are and PEG (mPEG-SVA) in freezer.
?. [Capping]
Remove all the liq. Wash again as before. Make sulfo-NHS-acetate solution: 20mg sulfo-NHS-acetate + 80 ul of 0.1M NaHCO3.Flow capping soln (Sulfo-NHS-acetate) 100 uL and leave it for 1 h in dark, RT.Sulfo-NHS-acetate in freezer.
?. [Cleaning]
Remove all liq. Wash as before. Fill the chamber with 1 mM MgCl2 soln in 1x PBS. Leave it in 4 C when not in use. MgCl2 on the shelve row 5.
?. [Testing]
NOTE: Do not leave the chamber in air. Always flow new solution to replace old one instead of emptying out the chamber. Use buffer (1 mM MgCl2 in 1x PBS) for imaging.Flow target solution (100 nM for test) into the chamber and incubate 15 mins. Then flow in buffer to remove target solution and image. To remove target bound to probes, wash with DI and leave it in DI for 5 mins.
TITLE: BSCI:414 Lab 6 and Lab 7--Analyze RT-qPCR Results from COVID-19 Test
AUTHORS: Harley King

[STEPS]
?. [Review Experiment]
Watch and discuss experimental video.https://youtu.be/9s5sACRqkZ8
?. [Review Results]
Download screenshots and "Human.sample.chart.results.JPG"  in our class folder, "Files>Lab Results>Lab 6 qPCR Data".
?. [Review Results]
Calculate the concentration of plasmid DNA used to generate the standards.7 plasmid standards were created.  ABC1TubeConcNotes21?Diluted 1ug plasmid DNA in 100ul H2O32?Diluted 10ul from tube 1 into 990ul H2O of tube 243?Diluted 100ul from tube 2 into 900ul H2O54?Diluted 100ul of tube 3 into 900ul H2O65?Diluted 100ul of tube 4 into 900ul H2O76?Diluted 100ul of tube 5 into 900ul H2O87?Diluted 100ul of tube 6 into 900ul H2OOur assay used the "nCoV-CDC-Control Plasmid" from Operon. The plasmid has been registered in Benchling.
ABC1TubeConcNotes21?Diluted 1ug plasmid DNA in 100ul H2O32?Diluted 10ul from tube 1 into 990ul H2O of tube 243?Diluted 100ul from tube 2 into 900ul H2O54?Diluted 100ul of tube 3 into 900ul H2O65?Diluted 100ul of tube 4 into 900ul H2O76?Diluted 100ul of tube 5 into 900ul H2O87?Diluted 100ul of tube 6 into 900ul H2O
TITLE: LB Media
AUTHORS: William Brydon, Sricharan Kadimi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">LB (Luria-Bertani) media is commonly used for bacterial culture. We use it for overnight cultures after transformations.</div></div>

[STEPS]
?. [Creating the Broth]
In a large glass bottle (at least 1L), add the following:Component                            AmountDeoinized Water                   Tryptone                                 Yeast Extract                         NaCl                                        (You do not have to add the exact amount of water, just make sure it is close to 950mL).
950 ml
10 g
5 g
10 g
?. [Creating the Broth]
Shake or stir to dissolve all the solutes.
?. [Sterlizing the Broth]
Autoclave bottle follwing Autoclave protocol on the liquid cycle. Choose the appropriate cycle for the amount of liquid you have. Make sure the bottle cap is on loosely
?. [Sterlizing the Broth]
Let bottle cool on the lab bench with loose cap
?. [Sterlizing the Broth]
Keep bottle in the cold room for storage, with the cap tightly closed. Make sure you keep the bottle STERILE. We want a clear solution and nothing growing in our broth
?. [Creating the Broth]
Put a stir bar into a 1L glass bottle and fill the glass bottle with 1L of DI water (using a graduated cylinder). Mark the level of the water with a thin Sharpie or label tape. Pour out ~50mL of the water.
We do this step because QSing the media in a graduated cylinder gets messy - it's often difficult to transfer the solute back and forth.
?. [Creating the Broth]
QS the solution to the mark you made in Step 1 (1L) using DI water.
?. [Creating the Broth]
Optional: We have never done this step, but it is recommended by the Sambrook Molecular Cloning manual.Adjust the pH of the solution to 7.0 using 5M NaOH (~0.2mL).
TITLE: 1M Potassium Phosphate Buffer, pH 6.0
AUTHORS: Adrien Assie, Buck Samuel

[DESCRIPTION]
Potassium Phosphate buffer recipe

[STEPS]
1. Start with700 mL

2. 108.3 g KH2PO4
3. 35.6 g K2HPO4
4. Adjust to 1000 mL

5. Autoclave
TITLE: High-throughput microbial metabarcoding amplification Protocol sequenced by DNBSEQ-G400
AUTHORS: Xiaohuan Sun, Yuehua  Hu, Zewei  Song

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used for bacteral 16S V4 and fungal ITS amplification following sequencing characterizaiton using DNBSEQ-G400 (previously known as BGISEQ-2000) sequencing platform.</div></div>

[STEPS]
?. Amplify the 16SV4 region and ITS region from samples using a pair of primers with unique barcodes. X refers to 6bp artificial DNA oligos as first-barcode. N refers to the 0-3 bases random nuclear acid, which used to shift sequences in template DNA to avoid synchronal signals caused by conserved DNA regions in amplicons while sequencing.  AB1The primer sequences2ITS7_with barcodeXXXXXXN(0-3)GTGARTCATCRARTYTTTG3ITS4_with barcodeXXXXXX N(0-3)CCTSCSCTTANTDATATGC4515F_with barcodeXXXXXXN(0-3)GTGYCAGCMGCCGCGGTAA5806R_with barcodeXXXXXXN(0-3)GGACTACNVGGGTWTCTAAT6ITS1Fngs_with barcodeXXXXXXN(0-3)GGTCATTTAGAGGAAGTAA7ITS2ngs_with barcodeXXXXXXN(0-3)TTYRCKRCGTTCTTCATCG
AB1The primer sequences2ITS7_with barcodeXXXXXXN(0-3)GTGARTCATCRARTYTTTG3ITS4_with barcodeXXXXXX N(0-3)CCTSCSCTTANTDATATGC4515F_with barcodeXXXXXXN(0-3)GTGYCAGCMGCCGCGGTAA5806R_with barcodeXXXXXXN(0-3)GGACTACNVGGGTWTCTAAT6ITS1Fngs_with barcodeXXXXXXN(0-3)GGTCATTTAGAGGAAGTAA7ITS2ngs_with barcodeXXXXXXN(0-3)TTYRCKRCGTTCTTCATCG
?. The PCR recipe was as follows:  of diluted template DNA,  of forward primer (10 M),  of reverse primer (10 M),  of nuclease-free water,  of 5X KAPA HiFi Buffer,  of KAPA dNTPs mix (10mM), and  of KAPA HiFi Polymerase. We amplified samples using the following cycling conditions:  for ; 30 cycles of  for ,  for , and  for ; and then a final extension at  for .
1 mL
1 mL
1 mL
14.5 mL
5 mL
1 mL
0.5 mL
98 °C
98 °C
55 °C
72 °C
72 °C
?. Run amplicons from each sample on an agarose gel. Note : Low-biomass samples may yield faint or no visible bands; alternative methods such as a Bioanalyzer could be used to verify presence of PCR product.
?. Quantify amplicons with invitrogen Qubit Fluorometric Quantitation dsDNA Assay Kit.Note: Follow manufacturer’s instructions.
?. Mix an equal amount () of amplicon from each sample with different barcodes into a single, sterile tube.
200 ng
?. Here showes a schematic representation to summarize the previous steps for amplification protocol. Sample DNA could be amplified in 96-well plate, each plate used a unique barcode ligated with ITS specific primers. Each PCR product with different barcode was pooled together, then ligated with second barcode and sequencing primers in order to make sequencing library. Then each library was pooled for sequencing.
TITLE: Lactophenol-aniline blue solution
AUTHORS: Andrea Sweigart

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Lactophenol-aniline blue recipe</div></div>

[STEPS]
?. [Make Lactophenol]
To make   of  stock Lactophenol:Combine:
100 ml
20 ml
40 ml
20 ml
20 ml
?. [Lactophenol-aniline blue recipe]
To prepare  working solution, combine: stock Lactophenol  to
100 ml
100 ml
1 ml
5 ml
TITLE: Protocols for &#34;Characterizing the diversity of enteric neurons using Dopamine Transporter (DAT)-Cre reporter mice&#34;.
AUTHORS: sherilyn.recinto, Shobina Premachandran, Sriparna Mukherjee, Adam Macdonald

[DESCRIPTION]
Dopaminergic neurons (DA) are the predominant cell type in the midbrain that synthesize dopamine, a neurotransmitter implicated in a number of behavioural processes, including motor function, the reward pathway, and satiety. In diseases affecting these neurons, such as in Parkinson’s disease (PD), there is growing evidence that the gut-brain axis and selective vulnerability of DA neurons plays a crucial role in disease. Most investigations relating to DA neurons in the gut rely on immunoreactivity to tyrosine hydroxylase (TH) - a rate-limiting enzyme in the production of dopamine. However, the reliability of TH staining as a marker of DA neurons has been questioned in recent years. Our aim is to perform a comprehensive characterization of DA neurons in the gut using a well-accepted reporter mouse line, expressing a fluorescent protein under the dopamine transporter promoter (DAT). Our findings confirm a unique localisation of DA neurons in the gut, and also unveil that there are discrete subtypes of DA neurons in the gut, which we characterised using both immunofluorescence and single cell transcriptomics. We observed distinct subtypes of DAT neurons expressing co-transmitters and modulators; some of them likely co-releasing acetylcholine, and a smaller population likely releasing nitric oxide; while others were positive for a slew of canonical DA markers (Vmat2, Girk2, Foxa2). Given the clear heterogeneity of DA gut neurons, further investigation is warranted to define their functional signatures and discover their inherent biological differences that put these cells at risk for neurodegeneration.

[STEPS]
TITLE: UC Davis - Lipoprotein profiling and partical size
AUTHORS: Peter Havel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:   </span></div><div class = "text-block">Lipoprotein profiling and Lipoprotein size will be provided for CM, VLDL, LDL and HDL as well as 20 additional subclasses of lipoprotein. Testing requires as little as10µL for full analytical testing.</div></div>

[STEPS]
?. The sample is loaded into a gel permeation column specifically designed for separating lipoprotein components. Then, lipoprotein is eluted from the column in order of larger particles to smaller ones.
?. Lipoprotein fractionated by particle size is fed into reaction coils, and the degraded products after reacting with reagents are sent to detectors. The levels of cholesterol and triglyceride are output in the form of chromatogram.
?. The data obtained is processed with our patented analyzing program. The final output is a composite chromatogram and numeric data on 4 major classes (CM, VLDL, LDL and HDL) and 20 subclasses.
TITLE: Do, Q. B. &amp; Nebuloni, F. et al. (2023) A fluid walled microfluidic platform for human neuronal microcircuits and axotomy.
AUTHORS: Quyen Do, Federico Nebuloni, Richard Wade-Martins

[DESCRIPTION]
This collection contains six protocols detailing methods used in Do, Q. B. & Nebuloni, F. et al. (2023) A fluid walled microfluidic platform for human neuronal microcircuits and axotomy.

[STEPS]
TITLE: Preparation of electrocompetent cells
AUTHORS: Shuning    Guo

[DESCRIPTION]
This protocol is used to prepare electrocompetent cells with high transformation efficiency.

[BEFORE_START]
All the medium and containers used in the protocol should be sterilized by high temperature autoclave.
All the steps that expose cells to air should be down in the super-clean worktable.

[STEPS]
1. Line the E. coli strain on a solid LB medium without resistance and culture at 37℃ for 12h.
 

2. Pick monoclonal cells from the medium and culture them in 5 ml LB medium at 37℃ for 12h.

 

3. Inoculate 100 ml of LB medium with 1% volume of E. coli culture from step 2.
 

4. Grow the cells at 37℃ shaking at 200 rpm to an OD600 of 0.4 (about 1h 20min).
 

5. Transfer the culture on the ice immediately and chill cells on ice for 30 min and chill the centrifuge at 4 ℃ at the same time.
6. 
 

7. Transfer the cells to two cold 50ml centrifuge bottles (45ml every bottle) and spin at 1000 x g for 15 min at 4℃.
8. Carefully pour off and discard the supernatant.
9. Gently resuspend the pellet in ice-cold ddH2O (43ml every bottle). Centrifuge at 1000 x g for 20 min at 4℃; carefully pour off and discard the supernatant.
10. Gently resuspend the pellet in ice-cold 10% glycerol (21ml every bottle). Centrifuge at 1000 x g for 20 min at 4℃; carefully pour off and discard the supernatant.
11. Gently resuspend the pellet in ice-cold 10% glycerol (858μl every bottle). Mix well and transfer the mixture in two bottles into on bottle. Centrifuge at 1000 x g for 20 min at 4℃; carefully pour off and discard the supernatant.
12. Gently resuspend the pellet in 172 μl ice-cold GYT medium. 
13. Transfer the suspension into ice-cold 1.5ml microfuge tubes (25μl every tube).
14. Freeze the suspension in -80℃ ice cuber for about 2h.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Tongcheng pigs (TC) and Yorkshire (YK) are two pig breeds with distinguished morphologies in muscle. A comprehensive study of porcine microRNAome (miRNAome) in longissimus muscle during 5 developmental stages (40, 55, 63, 70 and 90 dpc (days post coitum)) using Solexa sequencing technology, was carried out in order to compare miRNA expression involved in the differentiated regulation of skeletal muscle development between the TC and YK breeds. Known miRNAs and novel miRNAs were identified by alignment and annotation. MiRNA expression profiles were normalized by transcript per million (TPM). Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were used to cluster the samples based on the expression values of miRNAs. The short time-series expression miner (STEM) was conducted in order to further explore the temporal expression characteristics of miRNAs in the TC and YK breeds. Then the analysis of differentially expressed (DE) miRNAs was performed. Functional annotation of the predicted miRNA targets was performed based on Gene Ontology slim database (GO-Slim) and Kyoto Encyclopedia of Genes and Genomes database (KEGG). Ssc-miR-499-5p is the only multipoint (no less than 4 time points)-DE miRNA with considerable expression level during the time periods investigated. Combining the target prediction and dual luciferase reporter assay, we validated DSTN was the target gene of ssc-miR-499-5p. The results are expected to facilitate the understanding of the differences of miRNAs in myogenesis between the TC and YK breed and provide implications for the improvement of meat quality in animal production.</p>

[BEFORE_START]
<p>All studies involving animals were conducted according to the relevant guidelines and regulations approved by the Ethics committee of Huazhong Agricultural University, Wuhan City, Hubei Province. All the experimental animal procedures were followed the Recommendations in the Hubei regulations for the Administration of Affairs Concerning Experimental Animals, 2005. All the animals were slaughtered after low voltage electrical stunning.</p>

[STEPS]
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TITLE: Updated QIAGEN PowerSoil protocol
AUTHORS: 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Adapted from manufacturer instructions for sand extraction for Nanopore sequencing.</div><div class = "text-block">https://www.qiagen.com/au/shop//sample-technologies/dneasy-powersoil-kit/#resources</div></div>

[STEPS]
?.
TITLE: 3: 30mer branch melting temperatures (SABER-FISH)
AUTHORS: Jocelyn Y. Kishi, Sylvain W. Lapan, Brian J Beliveau, Emma R. West, Allen Zhu, Hiroshi M. Sasaki, Sinem Saka, Yu Wang, Constance L Cepko, Peng Yin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the design of 30mer branch sequences.</div></div>

[STEPS]
?. It's recommended to use a temperature at least 1 degree lower than the lowest melting temperature of all branch sequences you plan to use (see plot of melting temperatures Fig. S2).You can find these melting temperature curves reported for each sequence, as well as those computed for 20mer imagers, 42mer barcode sequences, and an example set of FISH probes, reported in (available on the Nature Methods website).