Dna purification kit

Oligonucleotide primers were obtained from Integrated DNA Technologies (IDT). Taq DNA polymerases were from Denville Scientific Inc. and Invitrogen. TRIzol reagent, SuperScript III Reverse Transcriptase, TOPO TA cloning kit, and GeneRacer Advanced RACE kit were from Invitrogen and dNTP were from New England BioLab Inc. Restriction enzymes were from New England BioLab Inc. and Promega. Plasmid miniprep kit, gel extraction kit, and DNA purification kit were from Zymo Research. Secondary antibodies with fluorescence for the Western blots were from Licor. IPP, DMAPP, GPP, FPP, and GGPP were from Sigma. [4-¹⁴C]isopentenyl diphosphate triammonimum salt (55.0 mCi/mml) was from PerkinElmer Life Sciences. Ubiquinone Q6 (UQ₆) and Q8 (UQ₈) were from Avanti Polar Lipids. Ubiquinone Q10 (UQ₁₀) was from Sigma-Aldrich. Silica gels HPTLC were from Analtech. All other reagents were analytical grade or better.

Briefly, approximately 2 × 10⁷ pooled HCASMC cells were fixed with 1% formaldehyde and quenched by glycine. The cells were washed three times with PBS and then harvested in ChIP lysis buffer (50 mM Tris-HCl, pH 8, 5 mM EDTA, 0.5% SDS). Crosslinked chromatin was sheared for 3 × 1 min by sonication (Branson SFX250 Sonifier) before extensive centrifugation. Four volumes of ChIP dilution buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100) was added to the supernatant. The resulting lysate was then incubated with Dynabeads™ Protein G (Thermo Scientific, 10009D) and antibodies at 4 °C overnight. Beads were washed once with buffer 1 (20 mM Tris pH 8, 2 mM EDTA, 150 mM NaCl, 1% Triton X100, 0.1% SDS), once with buffer 2 (10 mM Tris pH 8, 1 mM EDTA, 500 mM NaCl, 1% Triton X100, 0.1% SDS), once with buffer 3 (10 mM Tris pH 8, 1 mM EDTA, 250 mM LiCl, 1% NP40, 1% sodium deoxycholate monohydrate), and twice with TE buffer. DNA was eluted by ChIP elution buffer (0.1 M NaHCO3, 1% SDS, 20 μg/mL proteinase K). The elution was incubated at 65 °C overnight, and DNA was extracted with DNA purification kit (Zymo D4013). Library was sequenced on the Illumina HiSeq X instrument to 400 million 150-bp paired-end reads.

Genomic DNA was extracted from mammalian cells using the Sigma GenElute kit or the Qiagen DNeasy Blood & Tissue Kits. PCRs were performed using genomic DNA as template, with primers listed in Supplementary Table S1 as per the manufacturer's directions. Subsequently, PCR product was purified using the Zymo DNA purification kit and sent for analysis by Sanger sequencing along with primers listed in Supplementary Table S1. The chromatograms were analyzed with the TIDE analysis web tool (https://tide.nki.nl/).^{34 (link)}

After elution of the chromatin from the beads, proteinase K (100 μg/ml) was added, and samples were incubated for 2 h at 55°C. DNA was purified from proteinase K–treated samples using a DNA purification kit following the manufacturer instructions (Zymo Research) and was subsequently analyzed either by running a 2% low melting agarose (APEX) gel or by an Agilent 2100 Bioanalyzer by using the DNA 1000 Kit.

An alanine mutation was introduced into 4V + S gene by quick-change PCR. The PCR conditions were as follows: ~200 ng parent template, 0.3 μM forward primer, 0.3 μM reverse primer, molecular biology grade water, and 1X Primestar Max. Nucleotide sequences for all primers are given in Supplementary section II A (see Supplementary Information). PCR was performed in a volume of 50 μL with the following procedure: 98 °C 10 s, (98 °C 10 s, 62 °C 15 s, 72 °C 220 s) for 16 cycles, 72 °C 5 min. The resulting 4 V + S + K79A plasmid was purified and eluted with 35 μL hot water. This product was then subjected to DPN1(1 μL) digestion in cut smart buffer at 37 °C for 1 h. The plasmid was then gel purified using a Zymo DNA purification kit and transformed into BL21(DE3) E. coli cells. Transformed cells were recovered using 750 μL SOC medium for 1 h at 37 °C. Cells were spread on LB kanamycin agar plates and incubated at 37 °C overnight. Single colonies were picked, cultured, and stored as glycerol stocks; the desired mutations were verified by Sanger sequencing.