Frozen tissue was processed to nuclei using the ‘Frankenstein’ protocol from protocols.io. Briefly, snap-frozen glioblastoma tissue was thawed on ice and minced sharply into <1-mm pieces. Next, 500 μl of chilled Nuclei EZ Lysis Buffer (Millipore Sigma, NUC-101, no. N3408) was added, and tissue was homogenized 10–20 times in a Dounce homogenizer. The homogenate was transferred to a 1.5-ml Eppendorf tube, and 1 ml of chilled Nuclei EZ Lysis Buffer was added. The homogenate was mixed gently with a wide-bore pipette and incubated for 5 min on ice. The homogenate was then filtered through a 70-μm mesh strainer and centrifuged at 500g for 5 min at 4 °C. Supernatant was removed, and nuclei were resuspended in 1.5 ml of Nuclei EZ Lysis Buffer and incubated for 5 min on ice. Nuclei were centrifuged at 500g for 5 min at 4 °C. After carefully removing the supernatant (pellet might be loose), nuclei were washed in wash buffer (1× PBS, 1.0% BSA and 0.2 U μl−1 of RNase Inhibitor). Nuclei were then centrifuged and resuspended in 1.4 ml of wash buffer for two additional washes. Nuclei were then filtered through a 40-μm mesh strainer. Intact nuclei were counted after counterstaining with Trypan blue in a standard cell counter.
Nuclei ez lysis buffer
Manufactured by Merck Group
Sourced in United States
The Nuclei EZ Lysis buffer is a solution designed for the extraction and isolation of nuclei from various cell types. The buffer facilitates the lysis of cell membranes while preserving the integrity of the nuclei, allowing for their efficient separation and further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Am2696, by Thermo Fisher Scientific (10 mentions)
Dounce homogenizer, by DWK Life Sciences (9 mentions)
Protease inhibitor, by Roche (8 mentions)
N2615, by Thermo Fisher Scientific (7 mentions)
Nuclei buffer, by Genomics Plc (4 mentions)
Chromium system, by 10x Genomics (3 mentions)
Novaseq 6000, by Illumina (3 mentions)
Nuclei ez lysis buffer, by Eppendorf (2 mentions)
Dounce homogenizer, by Merck Group (2 mentions)
Rnase inhibitor am2696, by Thermo Fisher Scientific (2 mentions)
Recombinant protein g agarose, by Thermo Fisher Scientific (2 mentions)
N2615, by Promega (2 mentions)
Kimble dounce tissue grinder, by Merck Group (2 mentions)
Nextseqmid output 2 150bp kit, by Illumina (2 mentions)
Facsaria 2, by BD (2 mentions)
40 μm strainer, by Thermo Fisher Scientific (2 mentions)
Nextseq 500 machine, by Illumina (2 mentions)
15 cm dishes, by Thermo Fisher Scientific (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Ampure xp beads purification, by Beckman Coulter (1 mentions)
55 protocols using nuclei ez lysis buffer
1
Nuclei Isolation from Frozen Glioblastoma Tissue
2
Isolating Nuclei from Frozen Glioblastoma Tissue
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Frozen tissue was processed to nuclei using the Frankenstein protocol from Protocols.io. Briefly, snap frozen glioblastoma tissue was thawed on ice and minced sharply into <1 mm portions. 500 l chilled Nuclei EZ Lysis Buffer (Millipore Sigma, NUC-101 #N3408) was added and tissue was homogenized 10-20 times in a Dounce homogenizer. The homogenate was transferred to a 1.5 ml Eppendorf tube and 1 mL chilled Nuclei EZ Lysis Buffer was added. The homogenate was mixed gently with a wide bore pipette and incubated for 5 minutes on ice. The homogenate was then filtered through a 70-m mesh strainer and centrifuged at 500g for 5 minutes at 4°C. Supernatant was removed and nuclei were resuspended in 1.5 mL Nuclei EZ Lysis Buffer and incubated for 5 minutes on ice. Nuclei were centrifuged at 500g for 5 min at 4°C. After carefully removing the supernatant, nuclei were washed in wash buffer (1x PBS, 1.0% BSA, 0.2 U/μl RNase inhibitor).
Nuclei were then centrifuged and resuspended in 1.4 ml wash buffer for two additional washes.
Nuclei were then filtered through a 40 m mesh strainer. Intact nuclei were counted after counterstaining with Trypan blue in a standard cell counter.
Nuclei were then centrifuged and resuspended in 1.4 ml wash buffer for two additional washes.
Nuclei were then filtered through a 40 m mesh strainer. Intact nuclei were counted after counterstaining with Trypan blue in a standard cell counter.
3
Nuclei Isolation from Frozen Glioblastoma
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Frozen tissue was processed to nuclei using the Frankenstein protocol from Protocols.io. Briefly, snap frozen glioblastoma tissue was thawed on ice and minced sharply into <1 mm portions. 500 μl chilled Nuclei EZ Lysis Buffer (Millipore Sigma, NUC-101 #N3408) was added and tissue was homogenized 10-20 times in a Dounce homogenizer. The homogenate was transferred to a 1.5 ml Eppendorf tube to which 1 mL chilled Nuclei EZ Lysis Buffer was added. The homogenate was mixed gently with a wide bore pipette and incubated for 5 min on ice. The homogenate was then filtered through a 70 μm mesh strainer and centrifuged at 500 g for 5 min at 4 °C. Supernatant was removed and nuclei were resuspended in 1.5 mL Nuclei EZ Lysis Buffer and incubated for 5 min on ice. Nuclei were centrifuged at 500 g for 5 min at 4 °C. After carefully removing the supernatant, nuclei were washed in wash buffer (1x PBS, 1.0% BSA, 0.2 U/μl RNase inhibitor). Nuclei were then centrifuged and resuspended in 1.4 ml wash buffer for two additional washes. Nuclei were then filtered through a 40 μm mesh strainer. Intact nuclei were counted after counterstaining with Trypan blue in a standard cell counter.
4
Isolation of Intact Nuclei from Frozen Brain Tissue
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Frozen human brain tissue was thawed on ice and minced sharply into <1 mm pieces. Next, 500μl of chilled Nuclei EZ Lysis Buffer (Millipore Sigma, NUC-101, no. N3408) was added, and the tissue was homogenized 10–20 times in a Dounce homogenizer. The homogenate was transferred to a 1.5-ml Eppendorf tube, and 1 ml of chilled Nuclei EZ Lysis Buffer was added. The homogenate was mixed gently with a wide-bore pipette and incubated for 5 min on ice. The homogenate was then filtered through a 70-μm mesh strainer and centrifuged at 500× g for 5 min at 4°C. The supernatant was removed, and the nuclei were resuspended in 1.5 ml of Nuclei EZ Lysis Buffer and incubated for 5 min on ice. The nuclei were centrifuged at 500× g for 5 min at 4°C. After carefully removing the supernatant (sometimes the pellet was loose), the nuclei were washed in wash buffer (1× PBS, 1.0% BSA and 0.2 U μl– 1 RNase inhibitor). The nuclei were then centrifuged and resuspended in 1.4 ml of wash buffer for two additional washes. The nuclei were then filtered through a 40-μm mesh strainer. Intact nuclei were counted after counterstaining with Trypan blue in a standard cell counter.
5
ATAC-seq Protocol for Chromatin Accessibility
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The cells were dissociated from the plates with Accutase (Sigma, A6964) for 3 minutes at 37°C. ATAC-seq samples and libraries were generated as described previously (Buenrostro et al., 2015 ) except the nuclei were prepared using 100 μl of ice cold Nuclei EZ lysis buffer (Sigma, N3408). The nuclei pellets were resuspended in 10 μl H2O and nuclei were counted. The tagmentation reaction was performed with 50 thousand nuclei and 2.5 μl of Tn5 transposase (0.5 μM) in 25 μl reaction volumes for 30 mins at 750 rpm at 37°C. The tagmented genomic DNA was purified by using miniElute Reaction Cleanup kits (QIAGEN, 28204) and eluted in 10.5 μl. The libraries were generated by 9 cycles of PCR reaction using adaptor primers (Nextera Index kit; Illumina, FC-121-1012) and NEBNext high fidelity 2x PCR master mix (NEB, M0541), followed by two-sided size selection by Ampure XP beads purification (0.4x reaction volume then 1.2x reaction volume; Beckman Coulter Agencourt, A63881). The typical yield is between 150-300 ng. The sequencing was performed on an Illumina Next-seq genome analyzer according to the manufacturer’s protocols.
6
Single-cell RNA-seq of Rat Cell Nuclei
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RIN14B cells were put on ice, washed with chilled PBS, and then lysed with chilled Nuclei EZ lysis buffer (Sigma-Aldrich, NUC-101). Single cells were isolated with a 40 µm filter and pelleted in a centrifuge for 8 min, 800 rcf, 4°C. The nuclei were resuspended using PBS with 1% BSA and counted using a hemocytometer with trypan blue viability dye. The nuclei were centrifuged and resuspended at an appropriate volume for the 10X Chromium system (10X Genomics). The nuclei were counted once more to check the number and quality before proceeding with 10X Chromium processing and library construction as per the manufacturer’s instructions. Next Gen sequencing with a Chromium V2 chemistry was carried out on an Illumina NextSeq 500. Illumina NextSeq 500 pre-mRNA sequencing data were aligned to the Rattus norvegicus genome using CellRanger. The data were then analyzed with Seurat V3.0 as described previously (Butler et al., 2018 (link)).
7
Single-Nucleus Extraction from Tissue
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Single-nucleus extraction from tissue was performed as previously described [55 (link)]. Briefly, engrafted colonic tissues were finely minced with a razor then transferred to a Dounce tissue homogenizer (Kimble Chase KT885300-0002) in 2 mL of ice-cold Nuclei EZ Lysis buffer (Sigma #N-3408) supplemented with protease inhibitor (Roche #5892791001) and RNase inhibitors (Promega #N2615, Thermo Fisher Scientific #AM2696). The tissue was ground 20–30 times with the loose pestle. The homogenate was filtered through a 200-μm cell strainer (pluriSelect #43-50200) then transferred back to the Dounce homogenizer, ground with the tight pestle 10–15 times. The homogenate was incubated on ice for 5 min with an additional 2 mL of lysis buffer, then filtered through a 40-μm cell strainer (pluriSelect #43-50040) and centrifuged at 500g for 5 min at 4 °C. The incubation and centrifugation steps were repeated one time, followed by resuspension Nuclei Suspension Buffer (1× PBS, 1% BSA, 0.1% RNase inhibitor) and filtering through a 5-μm cell strainer (pluriSelect #43-50005). The nuclei were then loaded onto the 10x Chromium Single Cell Platform for encapsulation and barcoding.
8
Nuclei Isolation from Frozen Tissues
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Nuclei were isolated from frozen paired tumor and normal brain tissue samples separately using the “Frankenstein” nucleus isolation protocol described previously13 (link). Briefly, ∼40 mg of frozen tissue was homogenized in chilled Nuclei EZ Lysis Buffer (Sigma–Aldrich, St. Louis, MO), and the homogenate was filtered using a 35 μm cell strainer (Falcon, #352235). The solution was centrifuged at 500 × g for 5 min at 4 °C in a benchtop centrifuge. Nuclei were resuspended in EZ lysis buffer, centrifuged again, and equilibrated to nuclei wash/resuspension buffer (1× phosphate-buffered saline, 1% bovine serum albumin (BSA), 0.2 U/μL RNase Inhibitor). The nucleus washing procedure was repeated three times, after which staining with DAPI (10 μg/mL) or propidium iodide (PI) was performed. After isolation, the nuclei were sorted using a Bio-Rad S3e Flow Cytometer (Bio-Rad) in 10X Genomics reverse transcription reagents (without enzymes) to ensure that the nuclei were free of debris.
9
Nuclei Isolation for snRNA-seq
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For snRNA-seq, nuclei were isolated with Nuclei EZ Lysis buffer (NUC-101; Sigma-Aldrich) supplemented with protease inhibitor (Roche) and RNase inhibitor AM2696, Life Technologies). Samples were homogenized using a Dounce homogenizer (885302-0002; Sigma-Aldrich) in 1 ml of ice-cold Nuclei EZ Lysis buffer, and incubated on ice for 2 min with an additional 1 ml of lysis buffer. The pellet was resuspended, washed with 4 ml of buffer. Following centrifugation, the pellet was resuspended in nuclei suspension buffer (1× PBS, 1% bovine serum albumin, 0.1% RNase inhibitor). For full details see Supplementary Methods .
10
STAU1 RNA Immunoprecipitation in HCT116
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HCT116 cells were cross-linked using 1500 μJ cm−2 and collected in cold PBS. Fifty millions cells per samples were subjected to nuclear/cytoplasmic fractionation using Nuclei EZ Lysis Buffer (Sigma), cytoplasmic fractions were collected and the volume adjusted in 2 × polysome buffer (Tris–Cl pH 7.4 20 mM, NaCl 200 mM, MgCl2 2.5 mM, Triton-X 1%, DTT 1 mM, protease inhibitor cocktail, Superase-In). Lysates were pre-cleared with yeast RNA (0.1 mg ml−1) and recombinant protein G agarose (Thermo Scientific) for 30 min at 4 °C. One per cent of the sample were used as input, the remaining samples incubated with STAU1 antibody (Proteintech, 14225-1-AP) or IgG from rabbit serum (I5006, Sigma) overnight at 4 °C, The RNA-antibody complexes were collected by incubation with Dynabeads protein G beads (Invitrogen). The samples were washed four times and Proteinase K treated before the RNA was extracted for qRT–PCR analysis.
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