Tagment dna buffer

A small subset of nuclei was stained with DAPI, and fluorescent nuclei were manually counted using a hemocytometer to determine their concentration; ∼50,000 nuclei were transferred to a new tube in 22.5 µl water and mixed with 2.5 µl Tagment DNA Enzyme I and 25 µl Tagment DNA Buffer (Illumina, catalog #20034198). The tn5 transposition reaction was conducted at 37°C for 30 min with 300 rpm mixing. After tagmentation, the samples were taken through column purification, amplification, and double-sided size selection (100-1000 bp) with AmpureXP beads (Beckman Coulter, catalog #A63881). To reduce amplification bias, we evaluated a side qPCR reaction for each sample to determine the optimal number of PCR cycles for each sample to reach one-third maximum intensity. To get initial sample quality estimates and balance sample representation for deep sequencing, each sample was sequenced at 1-3 million reads on the Illumina Miseq system. Then, samples were pooled and paired-end sequenced on a Novaseq 6000 to ∼25 million unique, nonmitochondrial reads per sample for analysis.

ATAC‐seq was conducted primarily following previous studies.²¹, ²² In our study, about 50 000 C2C12 in the proliferation and differentiation (60 hours) were lysed in 50 μL ATAC‐seq lysis buffer (10 mmol/L Tris‐HCl, pH 7.4, 10 mmol/L NaCl, 3 mmol/L MgCl₂, 0.1% NP40, 0.1% Tween‐20 and 0.01% digitonin) and incubated on ice for 5 minutes. Add 1 mL cold ATAC‐buffer (10 mmol/L Tris‐HCl, pH 7.4, 10 mmol/L NaCl, 3 mmol/L MgCl₂ and 0.1% Tween‐20) was added to stop the lysis. The cells were further centrifuged at 500 RCF for 10 minutes at 4°C to collect the cell pellet after removing the supernatant. The following transposition reaction mix (25 µL Illumina Tagment DNA buffer Cat#: 15027866, 16.5 µL 1 × PBS, 0.5 µL 1% digitonin, 0.5 μL 10% Tween‐20, 2.5 μL ddH₂O and 5 μL Illumina Tagment DNA enzyme (Cat#: 15027916) was added to the cell pellet and incubated at 37°C for 1 hour on thermocycler. ZYMO RESEARCH DNA Clean & Concentrator‐5 Kit (Cat#: D4013) was used to purify the transposed DNA. Library preparation was following the instruction of previous study.²¹ Final library was electrophoresed in a 2% high‐resolution agarose gel and 100‐1000 bp fragments were cut out and sequenced by using Illumina HiSeq X Ten PE150 platform. The information of ATAC‐seq data is shown in Table S1 and Figure S1.

Approximately 100,000 iMG were pelleted at 300g and 4 °C. Next, pellets were gently resuspended in ice-cold 50 μl of lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1% IGEPAL CA-630) and spun down at 500g for 10 min and 4 °C. The supernatants were discarded and pellets gently resuspended in 50 μl of transposition reaction mix (25 μl of tagment DNA buffer (Nextera, Illumina), 2.5 μl of tagment DNA enzyme (Nextera, Illumina), 22.5 μl of nuclease free water) and incubated at 37 °C for 30 min. Tagmented DNA was purified using MinElute PCR purification kit (Qiagen) and size selected for 70–500 base pairs (bp) using AmpureXP beads (Beckman Coulter). Libraries were constructed and amplified using 1.25 μM Nextera index primers and NEBNext High-Fidelity 2× PCR Master Mix (New England BioLabs). A quantitative PCR was run to determine the optimal number of cycles. Libraries were gel size selected for 165–300 bp fragments and single-end sequenced for 100 cycles (PE100) on an Illumina NovaSeq 6000.

50,000 FACS isolated classical monocytes or NK cells were lysed in 50 μl lysis buffer (10 mM Tris-HCl ph 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL, CA-630, in water) on ice and nuclei were pelleted by centrifugation at 500 RCF for 10 min. Nuclei were then resuspended in 50 μl transposase reaction mix (1x Tagment DNA buffer (Illumina 15027866), 2.5 μl Tagment DNA enzyme I (Illumina 15027865), in water) and incubated at 37°C for 30 min on a PCR cycler. DNA was then purified with Zymo ChIP DNA concentrator columns (Zymo Research D5205) and eluted with 10 μl of elution buffer. DNA was then amplified with PCR mix (1.25 μM Nextera primer 1, 1.25 μM Nextera index primer 2-bar code, 0.6x SYBR Green I (Life Technologies, S7563), 1x NEBNext High-Fidelity 2x PCR MasterMix, (NEBM0541)) for 8–12 cycles, size selected for fragments (160–290 bp) by gel extraction (10% TBE gels, Life Technologies EC62752BOX) and single-end sequenced for 51 cycles on a HiSeq 4000 or NextSeq 500.