Searching the RRID Resource Information Network
Are you sure you want to leave this community? Leaving the community will revoke any permissions you have been granted in this community.
Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community
Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. Preparation of tissue culture plates for neural rosettes and neural progenitors. protocols.io dx.doi.org/10.17504/protocols.io.x85fry6 Copy
Authors: Ethan Lippmann, Hannah Wilson, Emma Neal
Group: Neurodegeneration Method Development Community
Summary: Human induced pluripotent stem cell (iPSC)-derived developmental lineages are key tools for in vitro mechanistic interrogations, drug discovery, and disease modeling. iPSCs have previously been differentiated to endothelial cells with blood-brain barrier (BBB) properties, as defined by high transendothelial electrical resistance (TEER), low passive permeability, and active transporter functions. Typical protocols use undefined components, which impart unacceptable variability on the differentiation process. We demonstrate that replacement of serum with fully defined components, from common medium supplements to a simple mixture of insulin, transferrin, and selenium, yields BBB endothelium with TEER in the range of 2,000-8,000 Ω × cm2across multiple iPSC lines, with appropriate marker expression and active transporters. The use of a fully defined medium vastly improves the consistency of differentiation, and co-culture of BBB endothelium with iPSC-derived astrocytes produces a robust in vitro neurovascular model. This defined differentiation scheme should broadly enable the use of human BBB endothelium for diverse applications.
Proper citation: Ethan Lippmann, Hannah Wilson, Emma Neal 2020. Protocol for Differentiation of Blood-Brain Barrier Endothelial Cells from Human Pluripotent Stem Cells. protocols.io dx.doi.org/10.17504/protocols.io.bd6ei9be Copy
Authors: Schuele Lab, Faria Zafar
Group: Neurodegeneration Method Development Community, Schuele lab_1
Summary: The purpose of this protocol is high-molecular weight DNA extraction from whole blood for genetic analyses, including no amplification long-range sequencing.
Proper citation: Schuele Lab, Faria Zafar 2020. DNA extraction from whole blood using simple salting out procedure. protocols.io dx.doi.org/10.17504/protocols.io.bbzqip5w Copy
Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community
Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. iPSC gDNA Extraction: For Screening Edited Clones. protocols.io dx.doi.org/10.17504/protocols.io.x8afrse Copy
Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community
Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. Sanger Sequencing. protocols.io dx.doi.org/10.17504/protocols.io.x8tfrwn Copy
Authors: Simone Codeluppi, Lars Borm, amit zeisel, Gioele La Manno, Josina A. van Lunteren, Camilla I. Svensson, Sten Linnarsson
Group: Human Cell Atlas Method Development Community, CZI Spatial Transcriptomics Protocol Repository, Neurodegeneration Method Development Community
Summary: osmFISH is a cyclic single molecule fluorescent in situ hybridization protocol used to quantify the expression level of specific transcripts in tissue sections by direct labeling of individual RNA molecules. The number of transcripts quantified in each round correspond the numbers of fluorophores available in the microscope setup. In order to quantify a large number of genes, osmFISH provides a method to remove the probes/labelling (stripping) from their targets and get the tissue ready for the next round of labeling - imaging - stripping.Even though the osmFISH protocol has been developed to perform multiple smFISH rounds, it can also be used as a quick and simple method for one-round smFISH. Furthermore, the protocol can be further extended to include more complex encoding/barcoding shemes that can be used to resolve a larger number of targets.
Proper citation: Simone Codeluppi, Lars Borm, amit zeisel, Gioele La Manno, Josina A. van Lunteren, Camilla I. Svensson, Sten Linnarsson 2018. osmFISH. protocols.io dx.doi.org/10.17504/protocols.io.psednbe Copy
Authors: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward
Group: Neurodegeneration Method Development Community
Summary: Transient protein expression can easily be studied in i3Neurons using lipid-based transfection. This protocol is identical to that in iPSCs (see Basic Protocol 2). i3Neurons are modestly transfectable, with 5 % to 10 % of cells showing fluorescent protein expression after 24 hr. We have found that refreshing neuronal medium 1 to 2 hr after transfection both allows successful DNA entry into cells and largely prevents cytotoxicity resulting from the transfection reagent. Unlike iPSCs, i3Neurons show increased protein expression/accumulation over time, with greater fluorescence 48 to 72 hr after transfection than at 24 hr. Transient transfections also show more durable expression in i3Neurons than iPSCs, likely because episomes are not diluted by cell division. i3Neurons can be transfected in suspension (i.e., re-plating after day 3 of differentiation) or as an adherent culture, although better results are observed in adherent cultures. They are also amenable to serial transfections (i.e., re-transfecting with the same construct 24 hr apart) if higher-percentage transfections are desired.
Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. TRANSFECTION OF i3NEURONS (Support Protocol 3) . protocols.io dx.doi.org/10.17504/protocols.io.5wug7ew Copy
Authors: Minjung Song, Florian Merkle
Group: Neurodegeneration Method Development Community
Summary: PurposeThis protocol describes image-based quantification of human pluripotent stem cells (hPSCs), which could be adapted for other cell populations that grow in a monolayer. It utilises a membrane-bound stain and requires a high-content imaging system such as a PerkinElmer Oprea Phenix or similar and downstream image analysis pipelines. The suggested dye labels live cells, fluoresces in far red, and shows no evidence of toxicity in hPSCs. This protocol is useful in situations where cell quantification by manual or automated counting becomes rate-limiting, such as when multiple lines are grown for high-throughput differentiation experiments on different genetic backgrounds, or for survival or morphology-based screens in systems where cells have clearly defined cell borders.
Proper citation: Minjung Song, Florian Merkle 2020. Live Cell Quantification using Image Analysis. protocols.io dx.doi.org/10.17504/protocols.io.bpmcmk2w Copy
Authors: Bruno Benitez
Group: Neurodegeneration Method Development Community, Functional Genomics
Summary: Protocol: Purification of nuclei from human brains using an iodixanol gradientUse BSL2 precautions. Samples are not known to be infective, but should be treated as such. Gloves, mask if outside hood, disposable gown. Every surface and reusable equipment (homogenizers, centrifuge tubes) washed with 25% bleach.Discarded supernatants or other tissue byproducts should go into 10% bleach for sterilization.Discarded tubes and tips go into biohazard waste stream.Solutions:Stock optiprep 60% Iodixanol [Sigma-Aldrich D1556-250mL]Dilutant:Final Concentrations: 900 mM KCL; 30 mM MgCl2; 120 mM Tricine-KOH (pH 7.8)Recipe for 50 mls1) 22.5 ml of 2M KCL, +2) 1.5 ml of 1M MgCl2, + 3) 6 ml of 1 M Tricine-KOH 4) brought up to 50 ml with molecular biology grade water. Components1) Tricine-KOH is 17.92 grams Tricine (Sigma T0377) and 100 mL RNAse free water for 1 M solution (pH to 7.8 with KOH as needed. Usually takes about 3-4 mL of 3M KOH).2) 1 M MgCl2 (AM9530G) and 2M KCl (AM9460G) are commercial RNAse free stocks from Ambion. Homogenization medium (need 6 ml per sample + ~ 2ml extra for 30% Iodixanol): Recipe (stable at 4C) for 100 mls / 50 mls1) .25 M Sucrose (8.56 g / 4.28g ) 2) 150 mM KCL (7.5 ml / 3.75of 2M)3) 5 mM MgCl2 (.5 ml / .25 ml of 1M)4) 20 mM Tricine-KOH (2 ml / 1 ml of 1M)5) ddH2O up to 100 ml /50 ml in RNAse free H20Supplementation (right before use): 1) For 4 samples make 35 mls of homogenization buffer and supplement with:1. 3.5 ul spermine, 2. 3.5 ul spermidine, 3. 35 ul RNASIN 4. EDTA free protease inhibitor tablet. Iodixanol Dilutions:50% Iodixanol 1) Recipe: 5 volumes stock (60%) optiprep + 1 volume of dilutant. 2) Scale: need 2 ml per sample tube + 3 ml per sample for 35% Iodixanol3) Example: 4 samples add 17.5 ml stock to 3.5 ml diluant.35% Iodixanol 1) Recipe: 7 ml 50% Iodixanol + 3 ml supplemented homogenization medium.2) Scale: need 4 ml per sample3) For 4 samples add 5.14 mL supplemented homogenization buffer to 12 mL of 50% Iodixanol.Resupsension Buffer for Nuclei Wash (new from 10x Genomics recipe)1) 22.5 ml of 2M KCL, +2) 1.5 ml of 1M MgCl2, + 3) 6 ml of 1 M Tricine-KOH 4) brought up to 50 ml with molecular biology grade water. Components1) Tricine-KOH is 17.92 grams Tricine (Sigma T0377) and 100 mL RNAse free water for 1 M solution (pH to 7.8 with KOH as needed. Usually takes about 3-4 mL of 3M KOH).2) 1 M MgCl2 (AM9530G) and 2M KCl (AM9460G) are commercial RNAse free stocks from Ambion. Homogenization medium (need 6 ml per sample + ~ 2ml extra for 30% Iodixanol): Recipe (stable at 4C) for 100 mls / 50 mls1) .25 M Sucrose (8.56 g / 4.28g ) 2) 150 mM KCL (7.5 ml / 3.75of 2M)3) 5 mM MgCl2 (.5 ml / .25 ml of 1M)4) 20 mM Tricine-KOH (2 ml / 1 ml of 1M)5) ddH2O up to 100 ml /50 ml in RNAse free H20Supplementation (right before use): 1) For 4 samples make 35 mls of homogenization buffer and supplement with:1. 3.5 ul spermine, 2. 3.5 ul spermidine, 3. 35 ul RNASIN 4. EDTA free protease inhibitor tablet. Iodixanol Dilutions:50% Iodixanol 1) Recipe: 5 volumes stock (60%) optiprep + 1 volume of dilutant. 2) Scale: need 2 ml per sample tube + 3 ml per sample for 35% Iodixanol3) Example: 4 samples add 17.5 ml stock to 3.5 ml diluant.35% Iodixanol 1) Recipe: 7 ml 50% Iodixanol + 3 ml supplemented homogenization medium.2) Scale: need 4 ml per sample3) For 4 samples add 5.14 mL supplemented homogenization buffer to 12 mL of 50% Iodixanol.Resupsension Buffer for Nuclei Wash (new from 10x Genomics recipe)1) Tricine-KOH is 17.92 grams Tricine (Sigma T0377) and 100 mL RNAse free water for 1 M solution (pH to 7.8 with KOH as needed. Usually takes about 3-4 mL of 3M KOH).2) 1 M MgCl2 (AM9530G) and 2M KCl (AM9460G) are commercial RNAse free stocks from Ambion.Homogenization medium (need 6 ml per sample + ~ 2ml extra for 30% Iodixanol): Recipe (stable at 4C) for 100 mls / 50 mls1) .25 M Sucrose (8.56 g / 4.28g ) 2) 150 mM KCL (7.5 ml / 3.75of 2M)3) 5 mM MgCl2 (.5 ml / .25 ml of 1M)4) 20 mM Tricine-KOH (2 ml / 1 ml of 1M)5) ddH2O up to 100 ml /50 ml in RNAse free H20Supplementation (right before use): 1) For 4 samples make 35 mls of homogenization buffer and supplement with:1. 3.5 ul spermine, 2. 3.5 ul spermidine, 3. 35 ul RNASIN 4. EDTA free protease inhibitor tablet. Iodixanol Dilutions:50% Iodixanol 1) Recipe: 5 volumes stock (60%) optiprep + 1 volume of dilutant. 2) Scale: need 2 ml per sample tube + 3 ml per sample for 35% Iodixanol3) Example: 4 samples add 17.5 ml stock to 3.5 ml diluant.35% Iodixanol 1) Recipe: 7 ml 50% Iodixanol + 3 ml supplemented homogenization medium.2) Scale: need 4 ml per sample3) For 4 samples add 5.14 mL supplemented homogenization buffer to 12 mL of 50% Iodixanol.Resupsension Buffer for Nuclei Wash (new from 10x Genomics recipe)1) .25 M Sucrose (8.56 g / 4.28g ) 2) 150 mM KCL (7.5 ml / 3.75of 2M)3) 5 mM MgCl2 (.5 ml / .25 ml of 1M)4) 20 mM Tricine-KOH (2 ml / 1 ml of 1M)5) ddH2O up to 100 ml /50 ml in RNAse free H20Supplementation (right before use): 1) For 4 samples make 35 mls of homogenization buffer and supplement with:1. 3.5 ul spermine, 2. 3.5 ul spermidine, 3. 35 ul RNASIN 4. EDTA free protease inhibitor tablet. Iodixanol Dilutions:50% Iodixanol 1) Recipe: 5 volumes stock (60%) optiprep + 1 volume of dilutant. 2) Scale: need 2 ml per sample tube + 3 ml per sample for 35% Iodixanol3) Example: 4 samples add 17.5 ml stock to 3.5 ml diluant.35% Iodixanol 1) Recipe: 7 ml 50% Iodixanol + 3 ml supplemented homogenization medium.2) Scale: need 4 ml per sample3) For 4 samples add 5.14 mL supplemented homogenization buffer to 12 mL of 50% Iodixanol.Resupsension Buffer for Nuclei Wash (new from 10x Genomics recipe)1) For 4 samples make 35 mls of homogenization buffer and supplement with:1. 3.5 ul spermine, 2. 3.5 ul spermidine, 3. 35 ul RNASIN 4. EDTA free protease inhibitor tablet. Iodixanol Dilutions:50% Iodixanol 1) Recipe: 5 volumes stock (60%) optiprep + 1 volume of dilutant. 2) Scale: need 2 ml per sample tube + 3 ml per sample for 35% Iodixanol3) Example: 4 samples add 17.5 ml stock to 3.5 ml diluant.35% Iodixanol 1) Recipe: 7 ml 50% Iodixanol + 3 ml supplemented homogenization medium.2) Scale: need 4 ml per sample3) For 4 samples add 5.14 mL supplemented homogenization buffer to 12 mL of 50% Iodixanol.Resupsension Buffer for Nuclei Wash (new from 10x Genomics recipe)Iodixanol Dilutions:50% Iodixanol 1) Recipe: 5 volumes stock (60%) optiprep + 1 volume of dilutant. 2) Scale: need 2 ml per sample tube + 3 ml per sample for 35% Iodixanol3) Example: 4 samples add 17.5 ml stock to 3.5 ml diluant.35% Iodixanol 1) Recipe: 7 ml 50% Iodixanol + 3 ml supplemented homogenization medium.2) Scale: need 4 ml per sample3) For 4 samples add 5.14 mL supplemented homogenization buffer to 12 mL of 50% Iodixanol.Resupsension Buffer for Nuclei Wash (new from 10x Genomics recipe)1) Recipe: 5 volumes stock (60%) optiprep + 1 volume of dilutant. 2) Scale: need 2 ml per sample tube + 3 ml per sample for 35% Iodixanol3) Example: 4 samples add 17.5 ml stock to 3.5 ml diluant.35% Iodixanol 1) Recipe: 7 ml 50% Iodixanol + 3 ml supplemented homogenization medium.2) Scale: need 4 ml per sample3) For 4 samples add 5.14 mL supplemented homogenization buffer to 12 mL of 50% Iodixanol.Resupsension Buffer for Nuclei Wash (new from 10x Genomics recipe)1) Recipe: 7 ml 50% Iodixanol + 3 ml supplemented homogenization medium.2) Scale: need 4 ml per sample3) For 4 samples add 5.14 mL supplemented homogenization buffer to 12 mL of 50% Iodixanol. i. 1x PBS with 1.0% BSA and 0.2 U/uL1) Recipe:___________ commercial PBS (46-013CM 10x PBS from Corning)2) Recipe: __________ BSA (001000162 from Jackson Immunoresearch)3) Recipe: ____1:1000______ RNAsinProtocol: 1. Turn on the Beckman Allegra 64R, and Eppendorf 5430R Centrifuges so they can get to 4o. i. For Beckman put in swing-out rotor (S0410).***Make sure acceleration and deceleration are set to 0 on Beckman*** 2. Supplement the homogenization medium. i. Add fresh .15mM spermine (1:10,000) (1 ul per 10 ml) ii. Add fresh .5mM spermidine (1:10,000) (1 ul per 10 ml) iii. Add 1 µl RNASIN per ml of homogenization medium if using for RNA iv. Add EDTA free protease inhibitor tablet 3. Prepare working concentrations of rest of solutions.4. Aliquot 2ml of supplemented homogenization medium per 15 ml homogenization tube for each brain. We have used 1g of tissue successfully to yield 15-20 million nuclei. We also used 2g and double that. 5. Check weight of brain sample and keep on ice in 2ml supplemented homogenization medium.6. Homogenize the brain tissue in the 2 ml supplemented homogenization medium. i. Be careful at all steps to treat all samples exactly equally with regard to handling, as different cell type’s nuclei might be more or less vulnerable to physical destruction. All pipetting and resuspension steps should be gentle and consistent to avoid shearing and clumping of nuclei. ii. Put tissue into 15 ml Kontes glass homogenizer tubes iii. Homogenize 10 strokes on ice with ‘A’, and 10 strokes on ice with ‘B’ pestle. i. Use in BSL2 space for human tissue (currently using chem fume hood, with diapers). iv. Check to see that no large pieces have escaped homogenization. v. Transfer pipette with 2ml strippette the homogenate equally into 2 x 2 ml siliconized snap cap microcentrifuge tubes. vi. Add another 500ul supplemented homogenization buffer to the glass homogenizer and transfer remaining homogenate to the same microcentrofuge tubes. i. After use with human tissue, decontaminate homogenizers with 10% bleach during a break in the protocol. Dispose of diapers, tips, etc, in BSL2 waste. ii. Then RNASe Zap, then rinse with ethanol then ddh20 water. 7. Spin tubes at low speed (500 RCF) in the Eppendorf for 5 minutes at 4o.8. Pour off the supernatant, add 1 ml supplemented homogenization medium, and turn over or flick to resuspend. i. Take 100 ul of supernatant and freeze at -80 if you want cytoplasmic RNA later9. Repeat step 8.10. While spinning you can add 4ml of 35% Iodixanol to a centrifuge tube for each sample. *Mix the solution up and down to mix again after it has been sitting* i. These are polycarbonate centrifuge tubes from Beckman Coulter, stored in cabinet under the Sorvall in the shared equipment room. ii. May need to do a quick spin of the 35% to get it to settle down if some is stuck on the walls.11. Resuspend each pellet in 700 uls of supplemented homogenization buffer. Combine all tubes for each sample into a single 15 ml conical. 12. Add an equal volume of 50% Iodixanol to the samples in the 15 ml conicals. This will create a final 25% Iodixanol. *Mix 50% Iodixanol right before usage*13. Pipette the samples up and down with low retention pipette tips and carefully layer the ~ 3ml-4mls of 25% Iodixanol (2 tubes) on top of the 4 mL of 35% Iodixanol using a low attachment tip (silver box). You will need to use the same volume of all samples so that tubes are balanced, so scale to your smallest sample. i. Layering is best done by holding the bottle almost horizontal and using a P1000 to pipette slowly into the wall of the tube slightly above the 35 % Iodixanol. It is important to make sure the layers do not mix. 14. Centrifuge the tubes at 10,000 rpm (top speed) for 30 minutes in the Beckman Allegra *Check acceleration and deceleration again*15. Carefully remove the centrifuge tubes. Take back to BSL2 space. The nuclei should be visible as a cloudy layer in the interface of the 25%/35% Iodixanol. with a transfer pippette, remove the top pink debris layer of myelin (suck off with a transfer pipette).16. With a low retention pipette remove the nuclei by vacuuming them up from just above the layer. Then place into a 1.5mL Eppendorf – NONSTICK TUBES (for nuclei). No more than 500 uls per tube. Usually 2 tubes per sample. 17. Add 1 ml of supplemented Nuclei Wash buffer to the tubes & mix. 18. Centrifuge at 500g’s (rcf) at 4o.19. Nuclei should be pelleted at the bottom20. Remove & discard supernatant 21. Resuspend nuclei in about 750 mls of Nuclei Wash buffer. Bring together all nuclei from the same sample into a single tube.22. Centrifuge at 500g’s (rcf) at 4o.23. Nuclei should be pelleted at the bottom24. Remove & discard supernatant 25. Resuspend nuclei in about 1000 mls of Nuclei Wash buffer26. Remove & discard supernatant 27. Resuspend nuclei in about 1000 ul of Nuclei Wash buffer. 28. Let it drip through a 40 um Nylon mesh cell strainer (#229481)to remove clumps per 10x protocol.29. Take out 20 uls, mix 1:1 with a 1:2000 DAPI solution, and count on hemacytometer or countess and confirm lack of clumps.30. Adjust aliquot of nuclei to desired dilution (1,200 cells per ul). Count again to confirm dilution.31. The rest may be stored as follows: Pellet and resuspend in 100% methanol
Proper citation: Bruno Benitez 2019. Single Nuclei extraction from frozen brain. protocols.io dx.doi.org/10.17504/protocols.io.2a5gag6 Copy
Authors: Jeffrey R. Moffitt, Xiaowei Zhuang
Group: Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community
Summary: Staining hundreds to thousands of RNA species each of which requires tens of unique encoding probes requires a very large number of unique oligonucleotide sequences. The sheer number of unique sequences makes traditional solid phase oligonucleotide synthesis prohibitively expensive in most cases. For example, assuming a modest cost of $0.10/base, an encoding probe length of ~100 nt, and the need for ~50,000 unique oligos for a single MERFISH measurement, the cost of the needed oligos would be $500,000! To circumvent this astronomical cost, we have developed a high-throughput approach to generating these probes which utilizes array-derived synthesis of complex oligonucleotide pools. These arrayderived complex oligonucleotide pools that contain ~100,000 custom designed sequences can be purchased for only a few thousand dollars. The challenge to using these pools is that each individual sequence is provided in quantities far too small to be used directly for labeling. Thus, we developed an enzymatic amplification protocol to generate the encoding probes in high quantity sufficient for RNA FISH experiments using the array-derived complex oligonucleotide pools as templates.
Proper citation: Jeffrey R. Moffitt, Xiaowei Zhuang 2018. RNA Imaging with MERFISH - Probe Construction. protocols.io dx.doi.org/10.17504/protocols.io.meqc3dw Copy
Authors: Jeffrey R. Moffitt, Xiaowei Zhuang
Group: Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community
Summary: The preparation and staining of samples for MERFISH follows closely the typical protocols used for smFISH (Raj et al., 2008). However, there are a few places in which we have modified these protocols to optimize MERFISH staining. Again, RNase contamination can destroy samples, so care should be taken to work in an RNase-free environment.
Proper citation: Jeffrey R. Moffitt, Xiaowei Zhuang 2018. RNA Imaging with MERFISH - Sample Preparation and Staining. protocols.io dx.doi.org/10.17504/protocols.io.merc3d6 Copy
Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community
Summary: All methods described in this collection should be performed using sterile technique.
Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. iPSC Cortical Differentiation. protocols.io dx.doi.org/10.17504/protocols.io.x9cfr2w Copy
Authors: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward
Group: Neurodegeneration Method Development Community
Summary: While transgenic populations may be enriched to near-purity using the techniques described above, the derivation of a clonal iPSC line descended from a single parent cell is necessary for proper genotyping and for many downstream applications, and it is thus standard practice following any genetic edit. One common method for isolating such clones, as described below, is by serial dilutions on two 6-well plates, followed by manual picking. As with all single-cell dissociations of iPSCs, the use of Y-27632 ROCK inhibitor is required until colonies are properly established; wells with fewer cells may require treatment for 2 to 3 days. 6-well plates are used because their wells have a larger surface area than other multiwell dishes, facilitating downstream picking. For highly enriched populations, serial dilutions may be reduced to one 6-well plate loaded with 1 × 105 cells in the first well, as there is a higher probability of identifying a purely positive colony.Following isolation, this protocol further describes basic genotyping in parallel with the gradual expansion of clonal lines, utilizing QuickExtract to prepare genomic DNA and PCR to test for the presence of a transgene of interest. Primer sequences and other specifics are included in more detail for the particular differentiation cassettes in Support Protocol 1. In general, basic genotyping by PCR should be performed as quickly as possible to screen out negative clones, while potentially positive clones should be confirmed by more stringent methods such as Sanger sequencing and western blotting as appropriate. To save culture reagents, negative clones may be immediately discarded, while all others should be expanded and frozen pending confirmation.
Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. ISOLATION AND VALIDATION OF CLONAL TRANSGENIC LINES (Basic Protocol 4). protocols.io dx.doi.org/10.17504/protocols.io.5f7g3rn Copy
Authors: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward
Group: Neurodegeneration Method Development Community
Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. ASTROCYTE PRODUCTION (Support Protocol 7.1). protocols.io dx.doi.org/10.17504/protocols.io.5xag7ie Copy
Authors: Tomasz Krzywkowski, Mats Nilsson
Group: Molecular Diagnostics - Mats Nilsson Group, Neurodegeneration Method Development Community
Summary: This protocol is for in situ detection of mRNAs and single nucleotide polymorphisms using padlock probes and rolling circle amplification. In the accompanying publication, we take advantage of a single nucleotide variant within conserved ACTB mRNA to successfully differentiate human and mice co-cultured cells and apply following protocol to genotype PCDH X and Y homologs in human brain tissue sections. These are used as examples in the accompanying publication and custom padlock probes can be designed to allow for the targeting of own desired mRNA. We provide a method for automated characterization and quantitation of target mRNA in single cells or chosen tissue area. mRNA of interest, harboring a polymorphism, is first reverse-transcribed to cDNA. Allele specific padlock probes are hybridized to the cDNA target and enzymatically circularized maintaining a physical link with the parent mRNA molecule. Lastly, circularized probes are replicated in situ, using rolling circle amplification mechanism to facilitate detection.This protocol is taken directly from the accompanying publication. Further details and background information can be found in the cited published article.
Proper citation: Tomasz Krzywkowski, Mats Nilsson 2019. Detection of single nucleotide polymorphisms with padlock probes. protocols.io dx.doi.org/10.17504/protocols.io.wjhfcj6 Copy
Authors: Daniel Gyllborg, Mats Nilsson
Group: Human Cell Atlas Method Development Community, Molecular Diagnostics - Mats Nilsson Group, Neurodegeneration Method Development Community
Summary: Protocol for multiplexed in situ sequencing in tissue sections as an image-based spatial transcriptomic method. This is the second iteration of In Situ Sequencing (HybISS: Hybridization based In Situ Sequencing) based on the principles published in Ke et al. Nature Methods, 2013 and more recently Qian et al. Nature Methods, 2019. Here we present a modified method in detection chemistry using sequencing by hybridization approach for combinatorial decoding. This results in a more robust method for detecting larger probe panels for a more high-throughput in situ sequencing method.
Proper citation: Daniel Gyllborg, Mats Nilsson 2020. HybISS: Hybridization-based In Situ Sequencing. protocols.io dx.doi.org/10.17504/protocols.io.xy4fpyw Copy
Authors: Andrea Argouarch
Group: Neurodegeneration Method Development Community
Summary: Detection of mycoplasma utilizing the bulldog-bio kit. Steps include, gDNA isolation from cells, nanodrop, PCR, and running an agarose gel to detect mycoplasma within a cell culture line. Image from https://cellculturedish.com/cell-culture-basics-mycoplasma-101-a-practical-guide-to-prevention-detection-and-elimination-of-mycoplasma-contamination/
Proper citation: Andrea Argouarch 2020. Mycoplasma. protocols.io dx.doi.org/10.17504/protocols.io.8gphtvn Copy
Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community
Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. Neural Rosette Formation and Selection. protocols.io dx.doi.org/10.17504/protocols.io.x86frze Copy
Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community
Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. Neural progenitor expansion. protocols.io dx.doi.org/10.17504/protocols.io.x88frzw Copy
Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community
Summary: Screening Edited iPSC ClonesExpanding and gDNA Extraction from iPSC ClonesNote: It takes approximately 1 week for iPSC picked into 96 well plates to be sufficiently confluent for freezing and screening. For screening purposes, a fraction of the cells picked into one well of a 96 well plate will be saved for DNA Extraction and the remaining will be kept in culture or frozen down.
Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. Screening Edited iPSC Clones. protocols.io dx.doi.org/10.17504/protocols.io.yazfsf6 Copy
Can't find your Protocol?
We recommend that you click next to the search bar to check some helpful tips on searches and refine your search firstly. If you want to find a specific protocol and you know the DOI of the protocol already, it's easier to enter a DOI to search. You can refine the search results using Facets on the left side of the search results page. If you are on the table view, you can also search in a specific column by clicking the column title and enter the keywords.
If you still could not find your protocol in the search results, please help us by adding it into the system — it's easy. Create and publish your protocols at Protocols.io.
Welcome to the dkNET Resources search. From here you can search through a compilation of resources used by dkNET and see how data is organized within our community.
You are currently on the Community Resources tab looking through categories and sources that dkNET has compiled. You can navigate through those categories from here or change to a different tab to execute your search through. Each tab gives a different perspective on data.
If you have an account on dkNET then you can log in from here to get additional features in dkNET such as Collections, Saved Searches, and managing Resources.
Here is the search term that is being executed, you can type in anything you want to search for. Some tips to help searching:
You can save any searches you perform for quick access to later from here.
We recognized your search term and included synonyms and inferred terms along side your term to help get the data you are looking for.
If you are logged into dkNET you can add data records to your collections to create custom spreadsheets across multiple sources of data.
Here are the sources that were queried against in your search that you can investigate further.
Here are the categories present within dkNET that you can filter your data on
Here are the subcategories present within this category that you can filter your data on
If you have any further questions please check out our FAQs Page to ask questions and see our tutorials. Click this button to view this tutorial again.
The NIDDK Information Network (dkNET) serves the needs of basic and clinical investigators by providing seamless access to large pools of data and research resources relevant to the mission of The National Institute of Diabetes Digestive and Kidney Diseases (NIDDK). dkNET is hosted at University of California San Diego, and is supported by NIH NIDDK grant U24DK097771.
Email: info_at_ dknet.org
