Beckman coulter ampure xp
DNA fragment size was verified on a 2100 Bioanalyzer Instrument using a High Sensitivity DNA Analysis Kit (both from Agilent Technologies Inc., Santa Clara, USA), and DNA concentration was verified using the Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies). DNA was separated by electrophoresis at 4V/cm for 2 hours at 4 ☌, and 200 to 400 bp fragments were excised and purified using the QIAquick Gel Extraction Kit (QIAGEN) as per the manufacturer’s instructions. PCR reactions were purified using the QIAquick PCR purification kit, eluted in 25 µL, mixed with 10 µL loading buffer (200 mg/mL Ficoll 400, 15 mg/mL Orange G and 2× Tris/Borate/EDTA buffer) and loaded on a 1.7% TBE agarose gel, prestained with SYBR safe DNA gel stain (Life Technologies). Each reactions was distributed between 4 PCR tubes and PCR was performed in a thermal cycler using the following program: initial denaturation at 98☌ for 30 seconds, amplification over 18 cycles of 98 ☌, 65 ☌ and 72 ☌ for respectively 10, 30 and 30 seconds, and a final extention at 72 ☌ for 7 min. To amplify these libraries, 50 µL Phusion High-Fidelity PCR Master Mix with HF Buffer (Thermo Scientific, Loughborough, UK) was added to each sample, together with primer 1 and 2 oligonucleotides to a final concentration of 500 nM. The latter mix was incubated at 37 ☌ for 30 min and at 95 ☌ for 5 min. These reactions were again purified using Agencourt AMPure XP (Beckman Coulter) and eluted in 50 µL water for ChIP sequencing, or 49 µL TE buffer and 1 µL USER enzyme mix (New England Biolabs) for nuclear RNA sequencing. Next, 4 µL 5× Ligation Buffer and 2 µL Quick Ligase (NEBnext Quick Ligation Module New England Biolabs) were added, and the reaction was incubated for 15 min at 37 ☌. This mix was incubated at 37 ☌ for 30 min, purified using Agencourt AMPure XP (Beckman Coulter) and eluted in 14 µL of pre-annealed barcoded adapters at a 20× molar amount relative to the estimated amount of dA-tailed DNA. The reaction was allowed to proceed for 2.5 hours at 16☌.Įnd repair or second strand synthesis reactions were purified using Agencourt AMPure XP (Beckman Coulter) and eluted in 15 µL dA-tailing mix (NEBNext dA-Tailing Module New England Biolabs).
cDNA-RNA duplexes were purified using 72 µL Agencourt AMPure XP (Beckman Coulter) and eluted in 20 µL of the following second-strand synthesis reaction mix: 0.33 mM dATP, dCTP and dGTP (Life Technologies), 0.33 mM dUTP (Sigma Aldrich), 0.25 U Ribonuclease H (Life Technologies) and 10 U DNA polymerase I (New England Biolabs). Water was added to make up the volume to 40 µL, and the sample was incubated at 25 ☌ for 5 min 50 ☌ for 30 minutes and inactivated at 70 ☌ for 15 min. Next 2 µL dNTP Mix (10 mM) (Life Technologies) were added, as well as 4 µl DTT (100 mM), 40U SUPERase.In, 400U SuperScript™ III Reverse Transcriptase and 8 µg Actinomycin D. This mixture was heated to 94 ☌ for 3 minutes for RNA fragmentation, and incubated on ice for at least 1 minute. RNA was subsequently reverse transcribed using SuperScript III Reverse Transcriptase (Life Technologies) as follows: 3 µg Random Primers (mostly hexamers Life Technologies) were added to 10 µL RNA and 8 µL 5× First-Strand Buffer. Genomic DNA was removed using the TURBO DNA-free kit (Life Technologies) as per the kit’s instructions.
Isolation of material from sorted nuclei: A solution of at least 100000 sorted nuclei was mixed with 3× its volume of TRIzol LS reagent (Life Technologies) and 2 µL glycogen (Life Technologies), and stored for at most one month at -80☌ until further processing according to manufacturer's instructions. Rats not able to fulfill the exercise protocol were withdrawn from the study.
Exercise training was performed over six weeks at a running speed of 15 m/min increasing with 2 m/min each week. Thereafter interval training was performed 6 days a week with 12 x 8 min intervals with 2 min resting period (6 m/min) after a warm-up period at 10 m/min. Hypertrophy induction: Male Sprague Dawley rats of ~280 g were assigned to interval training on a treadmill (Columbus Instruments, OH, US) with 25 degree inclination, with 5 days of acclimatization with running velocity for 6 m/min for 30, 45, 60, 75, 90 and 120 min. GEO help: Mouse over screen elements for information.Ĭell type: PCM1-positive nuclei from left ventricle of the heart