ATAC-seq analyses of sorted liver and lung ILC2s were performed as before, with minor modifications52 (link). A total of 5,000 − 50,000 cells were pelleted and washed with 50 µL of 1× PBS before treatment with 50 µL of 2× lysis buffer. The nuclei were resuspended in 40 µL of Tagment DNA enzyme buffer supplemented with 2.5 µL of Tn5 transposase (Illumina, 15027865) to tag and fragment the accessible chromatin. The mixture was incubated at 37 °C for 30 min. The DNA fragments were purified with a MinElute kit (Qiagen, 28004) and amplified by 11−13 PCR cycles based on the amplification curve. The libraries were sequenced for 60 cycles (single-end reads) on an Illumina HiSeq 1500 instrument. The reads were mapped to the mouse genome assembly version from December 2011 (GRCm38/mm10) by using the Bowtie2 mapping tool with a default setting. Peaks were called using MACS2 software with the broad option, and those identified in both replicates were used for further analyses (Fig. 7c ). Data quantification was conducted using the “annotatePeaks.pl” routine in HOMER. DARs were detected using the “getDifferentialPeaksReplicates.pl” routine in HOMER.
Tagment dna enzyme buffer
Manufactured by Illumina
The Tagment DNA enzyme buffer is a core component used in Illumina's DNA library preparation workflow. It functions to enzymatically 'tagment' or fragment DNA samples, preparing them for downstream sequencing analysis. The buffer provides the necessary reaction conditions for the Tn5 transposase enzyme to bind and insert adapter sequences into the DNA fragments in a controlled manner.
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2 protocols using tagment dna enzyme buffer
1
ATAC-seq Analysis of Liver and Lung ILC2s
2
ATAC-seq Protocol for Accessible Chromatin Analysis
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ATAC-seq was performed as previously described with minor modification (Buenrostro et al., 2013 (link); Shih et al., 2016 (link)). 50,000 cells were pelleted and washed with 50 µl of 1× PBS, followed by treatment with 50 µl of lysis buffer. The nuclei were resuspended in 40 µl of Tagment DNA enzyme buffer with 2 µl of Tn5 transposase (15027865, 15027866; Illumina) to tag and fragmentize the accessible chromatin. The reaction was incubated at 37°C with shaking at 300 revolutions per minute for 30 min. The fragmentized DNAs were purified using a QIAGEN MinElute kit and amplified with 11 or 12 PCR cycles, based on the amplification curve. The libraries were sequenced for 50 cycles (paired-end reads) on an Illumina HiSeq 1500.
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