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Digitonin

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Digitonin is a natural extract from the plant Digitalis purpurea. It is a non-ionic detergent used to permeabilize cell membranes in various laboratory applications.

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Lab products found in correlation

Tween 20, by Merck Group (10 mentions) Minelute reaction cleanup kit, by Qiagen (9 mentions) Np 40, by Merck Group (9 mentions) Minelute pcr purification kit, by Qiagen (9 mentions) Td buffer, by Illumina (6 mentions) Tn5 transposase, by Illumina (5 mentions) Tagment dna buffer, by Illumina (4 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (4 mentions) Qubit fluorometer, by Thermo Fisher Scientific (3 mentions) Hiseq 3000 4000, by Illumina (3 mentions) Minelute kit, by Qiagen (3 mentions) Nextseq 500 550 high output kit v2 75 cycle, by Illumina (3 mentions) Ampure xp bead, by Beckman Coulter (3 mentions) Igepal ca 630, by Merck Group (3 mentions) Ampure bead, by Beckman Coulter (2 mentions) Nextera dna library prep kit, by Illumina (2 mentions) Nebnext high fidelity 2x pcr master mix, by New England Biolabs (2 mentions) Tapestation, by Agilent Technologies (2 mentions) Nebnext 2 pcr master mix, by New England Biolabs (2 mentions) Novaseq sequencing system, by Illumina (2 mentions)

Close spelling variants of this product

Digitonin 1% (2 citations)

52 protocols using digitonin

1

Fluorescence Protease Protection Assay

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Fluorescence protease protection assays were performed as originally described (15 (link)) with the following modifications: HEK293 cells expressing indicated constructs were washed 1× in KHM buffer (110 mM potassium acetate, 3 mM MgCl2, 20 mM HEPES pH 7.2) and subsequently permeabilized with 80 μg/ml Digitonin (Promega) in KHM buffer for 90 s, followed by incubation with 50 μg/ml Proteinase K. Live confocal imaging was performed as described above, capturing 1-s intervals upon the addition of Digitonin and continued for the remainder of the time course.
Rudd J.C., Maity S., Grunkemeyer J.A., Snyder J.C., Lovas S, & Hansen L.A. (2023). Membrane structure and internalization dynamics of human Flower isoforms hFWE3 and hFWE4 indicate a conserved endocytic role for hFWE4. The Journal of Biological Chemistry, 299(8), 104945.
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2

ATAC-seq chromatin accessibility protocol

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ATAC-seq was performed as previously described15 (link), 38 (link), with minor adaptations. In each experiment, a maximum of 50,000 sort-purified cells were collected at 300 g for 5 min at 4 °C. After centrifugation, the pellet was carefully resuspended in the transposase reaction mix (12.5 μl 2xTD buffer, 2 μl TDE1 (Illumina), 10.25 μl nuclease-free water, and 0.25 μl 1% digitonin (Promega)) for 30 min at 37 °C. Following DNA purification with the MinElute kit eluting in 11 μl, 1 μl of eluted DNA was used in a quantitative PCR (qPCR) reaction to estimate the optimum number of amplification cycles. The remaining 10 μl of each library were amplified for the number of cycles corresponding to the Cq value (i.e., the cycle number at which fluorescence has increased above background levels) from the qPCR. Library amplification was followed by SPRI (Beckman Coulter) size selection to exclude fragments larger than 1,200 bp. DNA concentration was measured with a Qubit fluorometer (Life Technologies). Library amplification was performed using custom Nextera primers15 (link). Libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq 3000/4000 platform and the 50-bp single-end configuration. Chromatin accessibility mapping by ATAC-seq was done in two biologically independent experiments. Sequencing statistics are provided in Supplementary Table 1.
Krausgruber T., Fortelny N., Fife-Gernedl V., Senekowitsch M., Schuster L.C., Lercher A., Nemc A., Schmidl C., Rendeiro A.F., Bergthaler A, & Bock C. (2020). Structural cells are key regulators of organ-specific immune response. Nature, 583(7815), 296-302.
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3

Fast-ATAC-seq for Chromatin Accessibility

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Cells for ATAC-seq were sorted as described above. The Fast-ATAC version of the ATAC-seq protocol was performed as described (Corces et al., 2016). Briefly, 10,000 cells per replicate (2 replicates per cell type) were resuspended in transposase reaction mixture (25 μL of 2X Tagmentation DNA Buffer [TD buffer; Illumina, Cat#FC-121–1030], 2.5 μL Tn5 enzyme [Illumina, Cat#FC-121–1030], 0.5 μL 1% digitonin [Promega Cat#G9441], and 22 μL water), incubated for 30 minutes at 37 oC. The tagged and transposed DNA fragments were purified using MinElute Reaction Cleanup kit (QIAgen, Cat#28204) and eluted in 11 μL elution buffer (from the MinElute kit) and amplified as described (Buenrostro et al., 2013). Libraries were sequenced to a depth of 12–23 million reads per replicate.
Chan C.K., Gulati G.S., Sinha R., Tompkins J.V., Lopez M., Carter A.C., Ransom R.C., Reinisch A., Wearda T., Murphy M., Brewer R.E., Koepke L.S., Marecic O., Manjunath A., Seo E.Y., Leavitt T., Lu W.J., Nguyen A., Conley S.D., Salhotra A., Ambrosi T.H., Borrelli M.R., Siebel T., Chan K., Schallmoser K., Seita J., Sahoo D., Goodnough H., Bishop J., Gardner M., Majeti R., Wan D.C., Goodman S., Weissman I.L., Chang H.Y, & Longaker M.T. (2018). Identification of the Human Skeletal Stem Cell. Cell, 175(1), 43-56.e21.
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4

FAST-ATAC protocol for primary T cells

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We used the FAST-ATAC protocol4 (link). Human primary T cells from female subjects (age 12–46) were isolated from blood by Ficoll, followed by flow sorting of live cell single lymphocytes, CD3+ CD4+. Cells were sorted into RPMI with 10% FBS and were immediately processed for ATAC-seq. 10,000–40,000 cells were sorted into RPMI 1640 containing 10% fetal bovine serum. The cells were centrifuged at 500 × g for 5 min at 4 °C. All of the supernatant was aspirated, ensuring that the pellet was not disturbed in the process. The pellet was then resuspended in the tagmentation reaction mix (25 μL 2X TD Buffer (Illumina, 15027866), 2.5 μL TD Enzyme (Illumina, 15038061), 0.5 μL 1% Digitonin (Promega, G9441), 22 μL H2O) and mixed at 300 RPMs at 37 °C for 30 min on an Eppendorf Thermomixer. Immediately after the incubation, samples were purified using a minElute kit (Qiagen, 28006), eluting in 10 μL. For library preparation, sequencing, and analysis, please see the Supplementary Methods.
Mouri K., Guo M.H., de Boer C.G., Lissner M.M., Harten I.A., Newby G.A., DeBerg H.A., Platt W.F., Gentili M., Liu D.R., Campbell D.J., Hacohen N., Tewhey R, & Ray J.P. (2022). Prioritization of autoimmune disease-associated genetic variants that perturb regulatory element activity in T cells. Nature genetics, 54(5), 603-612.
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5

ATAC-seq Protocol for Profiling Chromatin Accessibility

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FACS purified cells were resuspended in 50 μl ATAC transposition reaction mix containing 25 μl 2x Tagment DNA Buffer (Illumina), 2.5 μl Tn5 transposase (Illumina), 0.5 µl 1% Digitonin (Promega #G9441) and incubated for 20-30 minute at 37°C with gentle agitation. For the M28Ag_1, M56Ag_1 and the CXCR5pos and CXCR5neg samples 16.5 μl PBS and 0.5 μl 10% Tween-20 were added to the transposition mix to reduce the level of background obtained in the sequencing as described by Corces et al. (32 (link)). DNA was purified using the MinElute Reaction Clean up kit (Qiagen #28204) before performing 5 cycles of PCR amplification using Nextera custom primers. The number of additional PCR cycles required to generate adequate material for sequencing was calculated using a qPCR side reaction as described (32 (link)). Amplified DNA was purified using Ampure Beads (Beckman Coulter) and libraries were validated by qPCR. Samples were sequenced on NextSeq® 500/550 High Output kit v2 75 cycles (Illumina, FC 404-2005) at the Genomics Birmingham sequencing facility. Two biological replicates were sequenced for each time point.
Bevington S.L., Fiancette R., Gajdasik D.W., Keane P., Soley J.K., Willis C.M., Coleman D.J., Withers D.R, & Cockerill P.N. (2021). Stable Epigenetic Programming of Effector and Central Memory CD4 T Cells Occurs Within 7 Days of Antigen Exposure In Vivo. Frontiers in Immunology, 12, 642807.
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6

ATAC-seq chromatin accessibility protocol

Cited 2 times
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ATAC-seq was performed as previously described15 (link), 38 (link), with minor adaptations. In each experiment, a maximum of 50,000 sort-purified cells were collected at 300 g for 5 min at 4 °C. After centrifugation, the pellet was carefully resuspended in the transposase reaction mix (12.5 μl 2xTD buffer, 2 μl TDE1 (Illumina), 10.25 μl nuclease-free water, and 0.25 μl 1% digitonin (Promega)) for 30 min at 37 °C. Following DNA purification with the MinElute kit eluting in 11 μl, 1 μl of eluted DNA was used in a quantitative PCR (qPCR) reaction to estimate the optimum number of amplification cycles. The remaining 10 μl of each library were amplified for the number of cycles corresponding to the Cq value (i.e., the cycle number at which fluorescence has increased above background levels) from the qPCR. Library amplification was followed by SPRI (Beckman Coulter) size selection to exclude fragments larger than 1,200 bp. DNA concentration was measured with a Qubit fluorometer (Life Technologies). Library amplification was performed using custom Nextera primers15 (link). Libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq 3000/4000 platform and the 50-bp single-end configuration. Chromatin accessibility mapping by ATAC-seq was done in two biologically independent experiments. Sequencing statistics are provided in Supplementary Table 1.
Krausgruber T., Fortelny N., Fife-Gernedl V., Senekowitsch M., Schuster L.C., Lercher A., Nemc A., Schmidl C., Rendeiro A.F., Bergthaler A, & Bock C. (2020). Structural cells are key regulators of organ-specific immune response. Nature, 583(7815), 296-302.
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7

Combinatorial barcoding snATAC-seq for human lung

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snATAC-seq using combinatorial barcoding (73 (link)) for human lung tissue was performed as described (72 (link)). In brief, pulverized tissue was suspended in nuclei permeabilization buffer (10 mM Tris-HCl [pH 7.5], 10 mM NaCl, 3 mM MgCl2, 0.1% Tween 20 [MilliporeSigma], 0.1% IGEPAL-CA630 [MilliporeSigma], and 0.01% Digitonin [Promega] in water; ref. 71 (link)) by pipetting, incubated for 10 minutes at 4°C, and filtered with a 30 μm filter (CellTrics). Nuclei were pelleted in a swinging bucket centrifuge (500g, 5 minutes, 4°C), resuspended in 500 μL high-salt tagmentation buffer (36.3 mM Tris-acetate [pH 7.8], 72.6 mM potassium-acetate, 11 mM Mg-acetate, 17.6% DMF), and counted using a hemocytometer. Two thousand nuclei were added to individual wells of a 96-well plate and tagmented with 1 μL barcoded Tn5 transposomes for 60 minutes at 37°C (74 (link)). After tagmentation, nuclei were combined, and 20 diploid nuclei were sorted per well into eight 96-well plates (total of 768 wells). Tagmented DNA was PCR amplified using primers with well-specific barcodes, and all wells were combined after completion of PCR. Purified and size-selected libraries were sequenced on a HiSeq4000 sequencer (Illumina) using custom sequencing primers with the following read lengths: 50 + 10 + 12 + 50 (Read1 + Index1 + Index2 + Read2).
Gally F., Sasse S.K., Kurche J.S., Gruca M.A., Cardwell J.H., Okamoto T., Chu H.W., Hou X., Poirion O.B., Buchanan J., Preissl S., Ren B., Colgan S.P., Dowell R.D., Yang I.V., Schwartz D.A, & Gerber A.N. (2021). The MUC5B-associated variant rs35705950 resides within an enhancer subject to lineage- and disease-dependent epigenetic remodeling. JCI Insight, 6(2), e144294.
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8

Characterization of C6 Rat Glioma Cells

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The C6 rat glioma cell line (accession number: CVCL 0194) was purchased from Riken Cell Bank (Wako, Japan). Roswell Park Memorial Institute Medium 1640 (RPMI 1640; catalog number (Cat#): 31800022) was obtained from Gibco (Grand Island, NY, USA). Tetramethylrhodamine ethyl ester (TMRE) (Cat#: T669), MitoSox Red (Cat#: M36008), and 2′,7′-Bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) acetoxymethyl ester (BCECF-AM) (Cat#: B1170) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). PBFI-AM (Cat#: P-1267) was purchased from Invitrogen (Carlsbad, CA, USA). CellTiter Glo (Cat#: G7571/2/3), BCA protein assay kit (Cat#: 23225), and digitonin (Cat#: 043-21376) were purchased from Promega (Fitchburg, WI, USA), Thermo Scientific (Rockford, IL, USA), and Sigma-Aldrich Co. (St. Louis, MO, USA), respectively. All other chemicals used were of the highest available purity.
Naima J, & Ohta Y. (2024). Potassium Ions Decrease Mitochondrial Matrix pH: Implications for ATP Production and Reactive Oxygen Species Generation. International Journal of Molecular Sciences, 25(2), 1233.
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9

ATAC-Seq of Activated T Cells

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ATAC-Seq was performed as previously described (Buenrostro et al., 2015 (link)), with some modifications. CD4 +T cells were activated with anti-CD3/CD28 in presence of IL-2 for 3 days. 50,000 cells were lysed and transpositions were performed using transposase mixture (Nextera DNA library prep kit, Illumina), supplemented with 0.01% digitonin (Promega; Madison, WI). Transposition reactions were incubated for 30 min at 37°C in a ThermoMixer (Eppendorf) with agitation at 300 rpm. DNA was purified using the MinElute Reaction Cleanup kit (Qiagen), and libraries amplified using NEBnext PCR master mix with the following primers:
Libraries were quantified using RT-qPCR prior to sequencing. All Fast-ATAC libraries were paired-end sequenced, 75 bp using a HiSeq2500 instrument. Quality of FASTQ files was performed using FASTX trimmer. More than 50 million reads were mapped, with <10% mapped, on average, to the mitochondrial genome. The reads were aligned to the hg19 (UCSC) version using Burrows-Wheeler Aligner (BWA-MEM). Peaks were called using MACS2 (Zhang et al., 2008 (link)) peak-caller, and the reads from input DNA sample were used as control. Visualization of the peaks was done using R Software.
Gonzalo-Gil E., Rapuano P.B., Ikediobi U., Leibowitz R., Mehta S., Coskun A.K., Porterfield J.Z., Lampkin T.D., Marconi V.C., Rimland D., Walker B.D., Deeks S, & Sutton R.E. (2019). Transcriptional down-regulation of ccr5 in a subset of HIV+ controllers and their family members. eLife, 8, e44360.
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10

ATAC-seq protocol for chromatin profiling

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ATAC-seq was performed as described previously [41 (link)]. In brief, 50,000 live cells were washed in cold PBS buffer and lysed in buffer with 0.01% Digitonin (Promega, G9441), 0.1% NP40 (Sigma, 11332473001) and 0.1% Tween-20 (Sigma, 11332465001). Transposition was performed at 37 °C, 1000 rpm for 30 min with Tn5 transposase (Vyzame, TD501). After purification of the DNA with Zymo DNA Clean and Concentrator 5 columns (Zymo Research, D4013), the material was pre-amplified for 5 cycles. Additional PCR cycles were evaluated by real-time PCR and final product was cleaned by AMPure XP Beads (Beckman, A63880) at a 1× ratio. HiSeqX10 sequencer (Illumina) was used to sequence the library with 150 bp paired-end reads mode at Annoroad Gene Technology Company (Beijing, China). Two biological replications were carried out for each time point.
Zhang J., Ouyang Z., Xia L., Wang Q., Zheng F., Xu K., Xing Y., Wei K., Shi S., Li C, & Yang J. (2023). Dynamic chromatin landscape encodes programs for perinatal transition of cardiomyocytes. Cell Death Discovery, 9, 11.
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