{"product_id":"plant-nuclei-isolation-kit-snrna-seq","title":"FireGene Universal Plant Nuclei Isolation Kit for Single-Nucleus Sequencing","description":"\u003cp\u003e\u003cstrong\u003eFireGene Universal Plant Nuclei Isolation Kit\u003c\/strong\u003e is designed for the isolation and extraction of cell nuclei from cryopreserved tissue samples of higher plants. The kit uses surfactants to release nuclei, remove chloroplasts, and maintain nuclear membrane stability. After filtration, centrifugation, density-layer separation, and chloroplast removal, the workflow generates clean single-nucleus suspensions suitable for single-nucleus transcriptome sequencing, single-cell ATAC sequencing, and related plant nuclear analysis experiments.\u003c\/p\u003e\n\u003ch2\u003eBackground Information\u003c\/h2\u003e\n\u003cp data-start=\"1267\" data-end=\"1583\"\u003ePlant tissues contain rigid cell walls, abundant chloroplasts, and diverse secondary metabolites, which can complicate nuclei preparation. For cryopreserved plant samples, nuclei isolation provides a useful approach for studying gene expression and chromatin accessibility when intact cell recovery is not practical.\u003c\/p\u003e\n\u003cp data-start=\"1585\" data-end=\"1801\"\u003e\u003cstrong data-start=\"1585\" data-end=\"1634\"\u003eFireGene Universal Plant Nuclei Isolation Kit\u003c\/strong\u003e is designed for frozen higher plant tissue samples and supports clean nuclei preparation through lysis, filtration, density-layer separation, and chloroplast removal.\u003c\/p\u003e\n\u003ch3 data-section-id=\"15x6sgr\" data-start=\"1803\" data-end=\"1821\"\u003eResearch Areas\u003c\/h3\u003e\n\u003cp data-start=\"1823\" data-end=\"1952\"\u003eThis kit is suitable for plant research fields focused on nuclear gene regulation, tissue heterogeneity, and molecular profiling.\u003c\/p\u003e\n\u003cul data-start=\"1954\" data-end=\"2245\"\u003e\n\u003cli data-section-id=\"1x9yxmc\" data-start=\"1954\" data-end=\"1979\"\u003ePlant molecular biology\u003c\/li\u003e\n\u003cli data-section-id=\"623sjb\" data-start=\"1980\" data-end=\"2007\"\u003ePlant functional genomics\u003c\/li\u003e\n\u003cli data-section-id=\"90febs\" data-start=\"2008\" data-end=\"2037\"\u003ePlant developmental biology\u003c\/li\u003e\n\u003cli data-section-id=\"1w5o8at\" data-start=\"2038\" data-end=\"2070\"\u003ePlant stress response research\u003c\/li\u003e\n\u003cli data-section-id=\"1603u2b\" data-start=\"2071\" data-end=\"2113\"\u003eCrop science and plant breeding research\u003c\/li\u003e\n\u003cli data-section-id=\"1yybilz\" data-start=\"2114\" data-end=\"2133\"\u003ePlant epigenetics\u003c\/li\u003e\n\u003cli data-section-id=\"1y48arh\" data-start=\"2134\" data-end=\"2167\"\u003ePlant single-nucleus multiomics\u003c\/li\u003e\n\u003cli data-section-id=\"1qvit7r\" data-start=\"2168\" data-end=\"2204\"\u003ePlant tissue heterogeneity studies\u003c\/li\u003e\n\u003cli data-section-id=\"41l8oe\" data-start=\"2205\" data-end=\"2245\"\u003eGene regulation and chromatin research\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003ch3 data-section-id=\"1fuj4ew\" data-start=\"2247\" data-end=\"2267\"\u003eKey Applications\u003c\/h3\u003e\n\u003cp data-start=\"2269\" data-end=\"2378\"\u003eThe prepared single-nucleus suspensions can be used in downstream workflows requiring clean nuclear material.\u003c\/p\u003e\n\u003cul data-start=\"2380\" data-end=\"2739\"\u003e\n\u003cli data-section-id=\"2haltg\" data-start=\"2380\" data-end=\"2425\"\u003eSingle-nucleus RNA sequencing, or snRNA-seq\u003c\/li\u003e\n\u003cli data-section-id=\"1bd39xk\" data-start=\"2426\" data-end=\"2470\"\u003eSingle-cell ATAC sequencing, or scATAC-seq\u003c\/li\u003e\n\u003cli data-section-id=\"1vdl0d5\" data-start=\"2471\" data-end=\"2503\"\u003eNuclear transcriptome analysis\u003c\/li\u003e\n\u003cli data-section-id=\"5n45lt\" data-start=\"2504\" data-end=\"2539\"\u003eChromatin accessibility profiling\u003c\/li\u003e\n\u003cli data-section-id=\"10w5ai\" data-start=\"2540\" data-end=\"2571\"\u003eCell-type composition studies\u003c\/li\u003e\n\u003cli data-section-id=\"1tyi7am\" data-start=\"2572\" data-end=\"2630\"\u003eGene expression analysis from cryopreserved plant tissue\u003c\/li\u003e\n\u003cli data-section-id=\"jgrmws\" data-start=\"2631\" data-end=\"2668\"\u003ePlant tissue heterogeneity analysis\u003c\/li\u003e\n\u003cli data-section-id=\"1j4qofn\" data-start=\"2669\" data-end=\"2739\"\u003eMolecular mechanism studies in plant development and stress response\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003ch2\u003eSpecifications\u003c\/h2\u003e\n\u003ctable\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth\u003eSpecification\u003c\/th\u003e\n\u003cth\u003eDetails\u003c\/th\u003e\n\u003c\/tr\u003e\n\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd\u003eProduct Name\u003c\/td\u003e\n\u003ctd\u003eUniversal Plant Nuclei Isolation Kit\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003eCatalog No.\u003c\/td\u003e\n\u003ctd\u003eFG-BA3319\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003eKit Size\u003c\/td\u003e\n\u003ctd\u003e10 reactions\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003eSample Type\u003c\/td\u003e\n\u003ctd\u003eHigher plant tissue\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003eCompatible Samples\u003c\/td\u003e\n\u003ctd\u003eCryopreserved plant tissue samples\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003eRecommended Starting Material\u003c\/td\u003e\n\u003ctd\u003eApproximately 200–300 mg frozen tissue\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003eMain Function\u003c\/td\u003e\n\u003ctd\u003eIsolation and extraction of plant cell nuclei\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003eWorkflow\u003c\/td\u003e\n\u003ctd\u003eLiquid nitrogen grinding, lysis, filtration, centrifugation, density-layer separation, chloroplast removal, nuclei resuspension\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003eDownstream Applications\u003c\/td\u003e\n\u003ctd\u003eSingle-nucleus transcriptome sequencing, single-cell ATAC sequencing, related nuclear analysis experiments\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003eRequired Instruments\u003c\/td\u003e\n\u003ctd\u003eHorizontal centrifuge, mortar and pestle, cell counter\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003eRequired Tissue Tools\u003c\/td\u003e\n\u003ctd\u003eSurgical scissors, surgical knives, forceps\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003eRequired Reagent\u003c\/td\u003e\n\u003ctd\u003eRNase inhibitor for nuclear RNA-related experiments\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003eRequired Consumables\u003c\/td\u003e\n\u003ctd\u003eZirconium beads, low-adsorption pipette tips, 5 mL and 15 mL centrifuge tubes, 70 μm cell strainers, optional 20 μm cell strainers\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003eProcessing Temperature\u003c\/td\u003e\n\u003ctd\u003eLow-temperature workflow; wet ice or ice blocks recommended\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003eStorage\u003c\/td\u003e\n\u003ctd\u003eMain reagents: 4°C, protected from light\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003eAdditive Storage\u003c\/td\u003e\n\u003ctd\u003eAdditive Solution ① and Additive Solution ②: -20°C, protected from light\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003eShelf Life\u003c\/td\u003e\n\u003ctd\u003eOne year under recommended storage conditions\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003eResearch Use\u003c\/td\u003e\n\u003ctd\u003eFor research use only\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003cp\u003e \u003c\/p\u003e\n\u003ch2\u003eKit Components\u003c\/h2\u003e\n\u003ctable style=\"width: 100%;\"\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth style=\"width: 52.5271%;\"\u003eComponent\u003c\/th\u003e\n\u003cth style=\"width: 26.3538%;\"\u003eCatalog Number\u003c\/th\u003e\n\u003cth align=\"right\" style=\"width: 20.2166%;\"\u003ePack Size\u003c\/th\u003e\n\u003c\/tr\u003e\n\u003c\/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 52.5271%;\"\u003eLysis Buffer\u003c\/td\u003e\n\u003ctd style=\"width: 26.3538%;\"\u003e\n\u003cmeta charset=\"utf-8\"\u003eFG-BA3319-A\u003c\/td\u003e\n\u003ctd align=\"right\" style=\"width: 20.2166%;\"\u003e20 mL\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 52.5271%;\"\u003ePre-Suspension Buffer\u003c\/td\u003e\n\u003ctd style=\"width: 26.3538%;\"\u003e\n\u003cmeta charset=\"utf-8\"\u003eFG-BA3319-B\u003c\/td\u003e\n\u003ctd align=\"right\" style=\"width: 20.2166%;\"\u003e15 mL\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 52.5271%;\"\u003ePost-Suspension Buffer\u003c\/td\u003e\n\u003ctd style=\"width: 26.3538%;\"\u003e\n\u003cmeta charset=\"utf-8\"\u003eFG-BA3319-C\u003c\/td\u003e\n\u003ctd align=\"right\" style=\"width: 20.2166%;\"\u003e50 mL\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 52.5271%;\"\u003ePB 1\u003c\/td\u003e\n\u003ctd style=\"width: 26.3538%;\"\u003e\n\u003cmeta charset=\"utf-8\"\u003eFG-BA3319-D\u003c\/td\u003e\n\u003ctd align=\"right\" style=\"width: 20.2166%;\"\u003e3 mL\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 52.5271%;\"\u003ePB 2\u003c\/td\u003e\n\u003ctd style=\"width: 26.3538%;\"\u003e\n\u003cmeta charset=\"utf-8\"\u003eFG-BA3319-E\u003c\/td\u003e\n\u003ctd align=\"right\" style=\"width: 20.2166%;\"\u003e6 mL\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 52.5271%;\"\u003ePB 3\u003c\/td\u003e\n\u003ctd style=\"width: 26.3538%;\"\u003e\n\u003cmeta charset=\"utf-8\"\u003eFG-BA3319-F\u003c\/td\u003e\n\u003ctd align=\"right\" style=\"width: 20.2166%;\"\u003e6 mL\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 52.5271%;\"\u003eCRS, Chloroplast Removal Solution\u003c\/td\u003e\n\u003ctd style=\"width: 26.3538%;\"\u003e\n\u003cmeta charset=\"utf-8\"\u003eFG-BA3319-G\u003c\/td\u003e\n\u003ctd align=\"right\" style=\"width: 20.2166%;\"\u003eNot specified\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 52.5271%;\"\u003eAdditive Solution ①\u003c\/td\u003e\n\u003ctd style=\"width: 26.3538%;\"\u003e\n\u003cmeta charset=\"utf-8\"\u003eFG-BA3319-H\u003c\/td\u003e\n\u003ctd align=\"right\" style=\"width: 20.2166%;\"\u003e4 mL\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 52.5271%;\"\u003eAdditive Solution ②\u003c\/td\u003e\n\u003ctd style=\"width: 26.3538%;\"\u003e\n\u003cmeta charset=\"utf-8\"\u003eFG-BA3319-I\u003c\/td\u003e\n\u003ctd align=\"right\" style=\"width: 20.2166%;\"\u003e150 μL\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003cp\u003e \u003c\/p\u003e\n\u003cp\u003e\u003cspan\u003e\u003cstrong\u003eProduct FAQ\u003c\/strong\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan\u003e\u003cstrong\u003e1.    Q: Is this kit only suitable for cryopreserved tissues of higher plants? Are there differences in nucleus extraction effects for different types of higher plant tissues (e.g., leaves, roots, stems)? Can it be used for fresh plant tissues or lower plant tissues (e.g., algae)?\u003c\/strong\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003cspan\u003eA: The kit is only suitable for cryopreserved tissues of higher plants and is not temporarily applicable to fresh plant tissues or lower plant tissues. Fresh plant tissues contain a large amount of water, which easily causes tissue adhesion during grinding and makes it impossible to form uniform fine powder. The nuclear membrane structure and fiber components of lower plants such as algae are significantly different from those of higher plants, and the kit reagents cannot stabilize the cell nuclei, easily leading to extraction failure. There are slight differences in the extraction effects of cryopreserved tissues from different parts of higher plants: leaf tissues (containing more chloroplasts) require extending the action time of chloroplast lysis buffer to 12 minutes; root and stem tissues have less chloroplast content, and an action time of 8 minutes is sufficient. Both can obtain high-purity cell nuclei with no significant difference in yield.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e \u003c\/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003cspan\u003e\u003cstrong\u003e2.    Q: The instruction manual requires weighing 150-200mg of frozen tissue. Will insufficient tissue quantity (e.g., 80mg) or excessive tissue quantity (e.g., 250mg) affect the nucleus extraction effect? How to adjust the operation?\u003c\/strong\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003cspan\u003eA: Yes, it will affect the effect. ① Insufficient tissue quantity (\u0026lt;120mg): The total number of cell nuclei is small, and the nuclear layer is not obvious during subsequent centrifugation and stratification, which easily leads to collection omission and a yield reduction of more than 50%. It is recommended to combine multiple 80mg tissue samples (to a total weight of 150mg) and follow the conventional steps to ensure the nuclear layer is clearly distinguishable. ② Excessive tissue quantity (\u0026gt;220mg): The space in the 5mL centrifuge tube is limited, and the lysis buffer cannot fully wrap the tissue fine powder, which easily leads to incomplete release of local cell nuclei and excessive local enzymolysis. The tissue needs to be processed in two tubes, with each tube containing 150-180mg of tissue and corresponding to 2mL of lysis buffer, to avoid the impact of tissue crowding on extraction efficiency.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e \u003c\/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003cspan\u003e\u003cstrong\u003e3.    Q: Step 2 requires grinding the frozen tissue into fine powder in a mortar pre-cooled with dry ice for 30 minutes. What impact will insufficient pre-cooling time of the mortar (e.g., 20 minutes) or thawing of the tissue during grinding have on the experiment?\u003c\/strong\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003cspan\u003eA: Insufficient pre-cooling of the mortar (\u0026lt;30 minutes) will cause the temperature of the mortar to rise during grinding, and the tissue is prone to thawing and adhesion, making it impossible to form uniform fine powder. Large tissue blocks are visible to the naked eye, and the release of cell nuclei during subsequent lysis is reduced by more than 40%. If the tissue thaws during grinding, plant cells will release a large number of enzymes due to the sudden temperature rise, damaging the nuclear membrane. After centrifugation, the nuclear layer is turbid, and the purity decreases by 60%. It is necessary to ensure that the mortar is pre-cooled for more than 30 minutes, and the tissue is kept in a frozen state throughout the grinding process. Dry ice can be added intermittently to maintain low temperature and prevent tissue thawing.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e \u003c\/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003cspan\u003e\u003cstrong\u003e4.    Q: Steps 4 and 17 use 70μm and 20μm cell sieves respectively. If the laboratory lacks one type of sieve, can it be replaced with a sieve of another pore size? For example, using 50μm instead of 70μm, or 10μm instead of 20μm?\u003c\/strong\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003cspan\u003eA: Random replacement is not allowed. ① The 70μm sieve is used to filter tissue debris after grinding. If replaced with a 50μm sieve, some cell nuclei will be retained (the diameter of higher plant cell nuclei is about 5-10μm; although they can pass through, the sieve is easily blocked by fiber debris, leading to filtration difficulties and even squeezing the cell nuclei to reduce activity). ② The 20μm sieve is used to remove impurities larger than cell nuclei. If replaced with a 10μm sieve, some cell nuclei will be retained, and the yield will decrease by 30%. If a 70μm sieve is lacking, extend the grinding time to 10 minutes to ensure the tissue fine powder is finer, then filter with an 80μm sieve (with an effect close to 70μm). If a 20μm sieve is lacking, let the resuspended nuclear suspension stand for 5 minutes after centrifugation, and aspirate the upper clearer liquid (avoiding bottom precipitated impurities) to minimize debris residue as much as possible.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e \u003c\/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003cspan\u003e\u003cstrong\u003e5.    Q: The centrifugation parameters for Steps 6, 10, and 13 are \"4℃, 500×g, 5 minutes\", \"4℃, 3000×g, 20 minutes\", and \"4℃, 700×g, 5 minutes\" respectively. What impact will deviations in centrifugation speed or time have? Can a vertical centrifuge replace a horizontal centrifuge?\u003c\/strong\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003cspan\u003eA: Deviations in centrifugation parameters have a significant impact: ① Step 6: If the speed is too low (\u0026lt;400×g), the cell nuclei cannot be fully precipitated and are discarded with the supernatant, resulting in a 40% yield reduction; if the speed is too high (\u0026gt;600×g), the centrifugal force is too strong, squeezing the cell nuclei and causing nuclear membrane rupture, with a 25% decrease in activity. ② Step 10: If the time is insufficient (\u0026lt;15 minutes), the solution stratification is unclear, and the nuclear layer is mixed with the impurity layer, making accurate collection impossible; if the speed is too low (\u0026lt;2800×g), the nuclear layer cannot settle at the interface of PB2 and PB3, resulting in a sharp 50% yield reduction. ③ Step 13: If the speed is too high (\u0026gt;800×g), the nuclear precipitate is compacted, making subsequent resuspension difficult. A vertical centrifuge cannot replace a horizontal centrifuge. The direction of centrifugal force of a vertical centrifuge is perpendicular to the centrifuge tube, which will cause disordered stratification of the solution and dispersion of the nuclear layer, making it impossible to form a clear three-layer structure. A horizontal centrifuge must be used.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e \u003c\/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003cspan\u003e\u003cstrong\u003e6.    Q: Step 2 mentions that \"lysis buffer, pre-suspension, and post-suspension need to be added with BSA at a final concentration of 1%\". What impact will deviations in BSA concentration (e.g., 0.5% or 2%) or forgetting to add BSA have on the cell nuclei?\u003c\/strong\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003cspan\u003eA: The core function of BSA is to protect the nuclear membrane and reduce mechanical damage during operation. ① Low concentration (0.5%): Insufficient protective effect, the nuclear membrane is prone to rupture during centrifugation and resuspension, a large number of nuclear fragments are visible under the microscope, and the purity decreases by 35%. ② High concentration (2%): Excessive BSA increases the solution viscosity, slows down the flow rate during subsequent filtration, and is easily mixed with cell nuclei, affecting downstream sequencing experiments (e.g., increasing non-specific signals). Forgetting to add BSA will cause the nuclear membrane to be directly exposed to the reagents, suffering double damage from mechanical force and chemical factors, with an activity reduction of more than 60%, making it unusable for subsequent experiments and requiring re-extraction.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e \u003c\/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003cspan\u003e\u003cstrong\u003e7.    Q: For nucleus RNA-related experiments, it is necessary to add RNase inhibitor at a final concentration of 1U\/μL to all reagents. How to specifically calculate the addition amount? What consequences will insufficient or excessive addition have?\u003c\/strong\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003cspan\u003eA: Calculation method for the addition amount: Taking \"per 1mL of reagent\" as the unit, if using 40U\/μL RNase inhibitor, 25μL needs to be added (1mL×1U\/μL÷40U\/μL=25μL). The lysis buffer needs to be additionally adjusted to a final concentration of 1.2U\/μL, that is, 30μL of 40U\/μL RNase inhibitor is added per 1mL of lysis buffer. Insufficient addition (\u0026lt;0.8U\/μL): It cannot effectively inhibit RNase activity, and the nuclear RNA will be degraded. The subsequent RNA extraction amount is less than 20% of the normal amount, and fragmentation is severe. Excessive addition (\u0026gt;1.5U\/μL): The preservative in the RNase inhibitor affects the activity of the cell nuclei, causing partial nuclear membrane rupture and a 20% decrease in purity. The calculated amount must be strictly added.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e \u003c\/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003cspan\u003e\u003cstrong\u003e8.    Q: Step 14 requires adding chloroplast lysis buffer and placing it on ice for 8-12 minutes, with \"the specific time depending on the amount of chloroplasts\". How to judge the amount of chloroplasts? What impact will insufficient or excessive action time have?\u003c\/strong\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003cspan\u003eA: Method for judging the amount of chloroplasts: If the ground tissue fine powder is dark green (e.g., leaf tissue), it indicates a large amount of chloroplasts; if it is light yellow or white (e.g., root tissue), it indicates a small amount of chloroplasts. Adjustment of action time: Dark green tissues require 10-12 minutes, light yellow\/white tissues require 8-10 minutes. During this period, observation can be made under a microscope every 2 minutes, and the process can be terminated when the green particles are significantly reduced. Insufficient action time (\u0026lt;8 minutes): Chloroplasts are not fully lysed, the precipitate is still green after centrifugation, and chloroplast fragments will interfere with nuclear detection in subsequent experiments (e.g., flow cytometry signal deviation). Excessive action time (\u0026gt;15 minutes): The chloroplast lysis buffer will damage the nuclear membrane, leading to a 30% decrease in nuclear activity. The time must be dynamically adjusted according to the color.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e \u003c\/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003cspan\u003e\u003cstrong\u003e9.    Q: When collecting the nuclear layer in Step 11, how to accurately identify the position of the nuclear layer? What impact will mistakenly aspirating the PB2 layer or debris layer have on subsequent experiments?\u003c\/strong\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003cspan\u003eA: After centrifugation, the solution is divided into three layers: the upper layer is the debris layer (turbid, approximately 1000μL), the middle layer is the nuclear layer (translucent, slightly milky white, approximately 400μL, located at the interface of PB2 and PB3), and the lower layer is the PB3 layer (transparent). During collection, a 1mL low-adhesion pipette tip should be used to slowly insert into the middle layer close to the liquid surface, and only the translucent liquid should be aspirated to avoid touching the upper and lower layers. Mistakenly aspirating the debris layer: A large amount of tissue debris is mixed into the nuclear suspension, causing significant impurity interference during subsequent counting and clogging the single-cell sequencing chip. Mistakenly aspirating the PB2 layer: The high-salt components in PB2 will damage the nuclear membrane, causing nuclear rupture, making it unusable for sequencing or staining experiments, and requiring re-centrifugation for collection.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e \u003c\/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003cspan\u003e\u003cstrong\u003e10.    Q: The kit needs to be stored at 4℃ in the dark with a validity period of 1 year. If the ice pack melts during transportation and the reagent is left at room temperature (25℃) for 1.5 hours, can it still be used? What consequences will long-term storage at room temperature have?\u003c\/strong\u003e\u003c\/span\u003e\u003cbr\u003e\u003cspan\u003eA: It can still be used after being left at room temperature for 1.5 hours, but it must be immediately returned to the 4℃ refrigerator and fully mixed before use. The surfactants and buffer components in the reagent have good stability during short-term room-temperature storage (≤2 hours), with an activity loss of ≤10%, which does not affect the extraction effect. Long-term storage at room temperature (exceeding 24 hours) will lead to: ① Decreased activity of the lysis buffer, inability to effectively release cell nuclei, and a 50% yield reduction; ② Inactivation of the chloroplast lysis buffer, inability to remove chloroplasts, and a sharp decrease in purity; ③ Changes in the pH value of the buffer, inability to stabilize the nuclear membrane, and easy rupture of cell nuclei during centrifugation. Finally, a qualified nuclear suspension cannot be obtained, and it cannot be used.\u003c\/span\u003e\u003c\/p\u003e","brand":"FireGene","offers":[{"title":"10 rxns","offer_id":47868382937300,"sku":"FG-BA3319-10","price":789.0,"currency_code":"USD","in_stock":true},{"title":"50 rxns","offer_id":47868382970068,"sku":"FG-BA3319-50","price":2759.0,"currency_code":"USD","in_stock":true}],"thumbnail_url":"\/\/cdn.shopify.com\/s\/files\/1\/0634\/0912\/7636\/files\/universal-preparation-of-nucleus-suspension-from-plant-tissuespng.png?v=1778574036","url":"https:\/\/firegene.com\/products\/plant-nuclei-isolation-kit-snrna-seq","provider":"FireGene","version":"1.0","type":"link"}