ATAC-Seq on MuSCs was performed based on the previously established OMNI-ATAC-Seq protocol (Corces et al., 2017 (link)). Briefly, five thousand MuSCs were sorted by FACS into 30 μL of the ATAC lysis buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM NaCl (Bioshop, 7647-14-5), 3 mM MgCl2 (Sigma, 7786-30-3), 0.1% Tween-20 (Sigma, P1379-1L), 0.1% NP-40 (Sigma, 74385), and 0.01% Digitonin (Promega, G9441) in a 0.2 mL microtube. Cells were incubated in the lysis buffer for 5 min on ice and then 3 min at room temperature (RT). Cells were then washed with 100 µL of wash buffer composed of 10 mM Tris-HCl (pH 7.5), 10 Mm NaCl, 3 mM MgCl2 and 0.1% Tween-20, and were centrifuged at 800 g for 10 min. The pellet was resuspended in 10 µL of transposition mixture (5 µL TD buffer, 3.2 µL PBS, 0.89 µL Tn5 (Illumina, 20034197), 0.1% Tween-20, 0.01% Digitonin and 0.75 µL nuclease free water). Transposition was performed for 20 min at 37 °C while shaking the tubes every 5–7 min. The DNA was then purified using a QIAquick PCR Purification Kit according to the manufacturer’s guidelines.
Digitonin
Manufactured by Illumina
Digitonin is a natural product derived from the plant Digitalis purpurea. It is a saponin glycoside with detergent-like properties that can be used to selectively permeabilize cell membranes. Digitonin is commonly used in biochemical and cell biology applications to facilitate the extraction or access of intracellular components for analysis.
Automatically generated - may contain errors
Lab products found in correlation
Np 40, by Merck Group (2 mentions)
Digitonin, by Promega (2 mentions)
Tn5 transposase, by Illumina (2 mentions)
Digitonin, by Merck Group (2 mentions)
Tagment dna buffer, by Illumina (2 mentions)
Nextseq instrument, by Illumina (1 mentions)
Minelute purification kit, by Qiagen (1 mentions)
Td buffer, by Illumina (1 mentions)
Bioanalyzer high sensitivity dna chip, by Agilent Technologies (1 mentions)
Facsaria flow sorter, by BD (1 mentions)
Tde tagmentation enzyme, by Illumina (1 mentions)
Ampure spri beads, by Beckman Coulter (1 mentions)
Np 40, by Roche (1 mentions)
Hiseq 4000, by Illumina (1 mentions)
Tween 20, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Mgcl2, by Merck Group (1 mentions)
Nextseq 500 550 high output kit v2 75 cycle, by Illumina (1 mentions)
Minelute reaction cleanup kit, by Qiagen (1 mentions)
Ampure bead, by Beckman Coulter (1 mentions)
5 protocols using digitonin
1
ATAC-Seq for Profiling Chromatin Accessibility in MuSCs
2
OMNI-ATAC-Seq for Single-Cell Genomics
3
scRNA-seq Library Preparation Protocol
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Infected cultures of CD4 T cells were stained with the viability dye Zombie Violet (ZV, Biolegend) and 50,000 live (ZV-) cells were sorted into 5mL polypropylene tubes using a FACSAria flowsorter (Becton Dickson) per replicate. The sorted cells were then washed with phosphate buffered saline (PBS) and resuspended in 50ul TD buffer (Illumina) with 0.01% digitonin and 2.5ul TDE Tagmentation enzyme (Illumina). The cells were then agitated in a thermomixer at 37C, 300rpm for 30mins to allow transposon-mediated tagmentation to occur. Tagmented DNA was then purified a Minelute purification kit (Qiagen). DNA fragments were PCR amplified using dual barcoded primers and re-purified using a Minelute kit followed by a 1.5x final cleanup step with Ampure SPRI beads (Agencourt). Libraries were quantified by Qubit DNA assay (Invitrogen) and size distribution of the library was determined using a High Sensitivity DNA BioAnalyzer chip (Agilent). Samples were sequenced using an Illimuna Hiseq2500 to a depth of 50-150M reads with 150bp paired end sequencing.
4
ATAC-seq Library Preparation Protocol
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ATAC-seq was performed as described in the published Omni-ATAC protocol (Corces et al., 2017 (link)). Briefly, 5,000–10,000 cells were sorted into FACS buffer and washed twice with 1 ml ice-cold ATAC-seq resuspension buffer (RSB; 10 mM Tris, pH 7.4 [Invitrogen], 10 mM NaCl [Millipore-Sigma], 3 mM MgCl2 [Millipore-Sigma]) by centrifugation at 0.5 ×g for 5 min at 4°C. After centrifugation, the supernatant was carefully removed to avoid the cell pellet, which was then gently resuspended in 50 µl ATAC-seq RSB containing 0.1% NP-40 (Roche), 0.1% Tween-20 (Millipore-Sigma), and 0.1% digitonin (Millipore-Sigma). The lysis reaction was incubated on ice for 3 min, 1 ml ATAC-seq RSB containing 0.1% Tween-20 was added, and the nuclei were centrifuged at 0.5 ×g for 10 min at 4°C. The supernatant was subsequently removed, and the pelleted nuclei were gently resuspended in 50 µl transposition mix (25 µl 2× Tagment DNA buffer [Illumina], 2.5 µl Tn5 transposase [Illumina], 16.5 µl 1× PBS, 0.5 µl 1% digitonin, 0.5 µl 1% Tween-20, and 5 µl water). Transposition reactions were incubated for 30 min in a 37°C water bath. Zymo DNA Clean and Concentrator kits were used to clean up the transposition reactions, and libraries were prepared as previously described (Buenrostro et al., 2015a (link)). Libraries were sequenced by 50 bp single-end, dual-index sequencing on an Illumina HiSeq 4000.
5
Omni ATAC-Seq for Profiling T Cell Epigenomes
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The omni ATAC‐Seq protocol was modified from Corces et al (2017 ). Briefly, 2W1S‐specific T cells were purified by flow cytometry from CD4creERT2 × Il7rf/f × ROSA26tdRFP mice (3,000–11,000 IL‐7Rα‐deficient cells) or from Il7rf/f × ROSA26tdRFP mice (8,000–20,000 IL‐7Rα+ cells). IL‐17a fate‐mapped Th17 cells were purified by flow cytometry from Il17aCre × ROSA26tdRFP+ mice (50,000 tdRFP+ cells). Cells were resuspended in 50 μl ATAC transposition reaction mix containing 25 μl 2× Tagment DNA Buffer (Illumina), 2.5 μl Tn5 transposase (Illumina), 16.5 μl PBS, 0.5 μl 10% Tween‐20, and 0.5 μl 1% Digitonin (Promega #G9441) and incubated for 20 min at 37°C. For the 2W1S‐specific Th1 memory cells, 40,000 cells were resuspended in 50 μl ATAC transposition reaction mix containing 25 μl 2× Tagment DNA Buffer (Illumina), 2.5 μl Tn5 transposase (Illumina), and 0.5 μl 1% Digitonin and incubated for 30 min at 37°C. DNA was purified using the MinElute Reaction Clean up kit (Qiagen #28204) before PCR amplification using Nextera custom primers. Optimal amplification was achieved by a qPCR side reaction as described (Corces et al, 2017 ). Amplified DNA was subsequently purified using Ampure Beads (Beckman Coulter) prior to validation. Libraries were sequenced on NextSeq® 500/550 High Output kit v2 75 cycles (Illumina, FC 404‐2005) at the Genomics Birmingham sequencing facility.
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