Micro bio spin p 30 gel column

For each population collection, total RNA was extracted from the antennae using Trizol (Invitrogen), followed by treatment with DNase (DNAfree, Ambion). The RNA sample from each collection was further purified using Micro Bio-Spin P-30 gel columns (BioRad). After quantification using an Agilent 2100 Bioanalyser the RNAs from the different collections in each of the four sampling sides were pooled. mRNA isolation and cDNA library preparation for each of the four samples were performed using the Illumina TruSeq RNA sample preparation kit (Illumina Inc., San Diego, CA, USA). The libraries were barcoded, pooled and sequenced as a 100 bp paired end run on one lane of an Illumina HiSeq 2000 platform at a concentration of 8 pM.

A single-stranded DNA sequence T7ɸ2.5-A-32 (5′-CAGTAATACGACTCACTATTAGGCCTCTCGCTCTGCTGGGTGTGCGCTTGC-3′), which contains a T7 ɸ2.5 promoter at the 5′-end and a unique adenosine at the transcription start site, and its reverse complementary sequence (5′-GCAAGCGCACACCCAGCAGAGCGAGAGGCCTAATAGTGAGTCGTATTACTG-3′) were synthesized and annealed with each other to get the double-stranded DNA (dsDNA) template. In vitro transcription was carried out at 37 °C overnight with the reaction (100 μL) containing 2 μg of the dsDNA template, 1 × T7 polymerase buffer (New England Biolabs), 1 mM CTP, 1 mM GTP, 1 mM UTP, and 1 mM ATP (for ppp-RNA) or NAD⁺ (for NAD-capped RNA) or FAD (for FAD-capped RNA) or dpCoA (for dpCoA-capped RNA) or ADPR (for ADPR-capped RNA) or Ap₄A (for Ap₄A-capped RNA), and 1 U/μL T7 RNA polymerase (New England Biolabs; M0251S). The RNA products were treated with DNase I (Roche) at 0.2 U/μL at 37 °C for 30 min, extracted by phenol/chloroform (5:1, pH 4.5), and then precipitated with ethanol. Unincorporated nucleotides were removed using Micro Bio-Spin P-30 Gel Columns (Bio-Rad) or by performing gel recovery after acryloylaminophenyl boronic acid affinity gel electrophoresis.

The FHV in vitro replication assay (IVRA) described here was adapted from Short et al. [36 (link)]. Briefly, 8 µL of CMP was added to a replication buffer (50 mM Tris, pH 8.0; 18 mM MgCl₂; 30 mM KCl; 16 mM NaCl; 250 mM sucrose; 10 µg/mL actinomycin D; 0.2 U/µL RiboLock; 1 mM ATP/GTP/UTP; 10 µM CTP; 5 µCi of CTP[α-32P] (specific activity, 3000 Ci/mmol, Perkin Elmer) to a final volume of 50 µL. The reaction was incubated for 90 min at 30 °C and the unincorporated nucleotides were removed using Micro Bio-Spin P-30 gel columns, Tris buffer, RNase-free (Bio-Rad, Berkeley, CA, USA). RNA isolation was performed as described by Scholte et al. [37 (link)]. In brief, acid phenol (Ambion) was used for the extraction following which RNA was precipitated with isopropanol, washed with 75% ethanol, and re-suspended in 20 µL of 1 mM sodium citrate (pH 6.4). A denaturing mixture (67% formamide, 23% formaldehyde, 6.7% glycerol, 13 mM MOPS (pH 7.2), 6.7 mM NaAc, 2.7 mM EDTA, 0.07% SDS, and 0.03% bromophenol blue) [37 (link)] was added to the RNA samples, incubated at 75 °C for 15 min, briefly vortexed and put on ice. RNAs were separated in a 1% denaturing formaldehyde agarose gel for 2 h at 100 V. The gel was dried and exposed to a phosphorimaging plate. Detection was performed using Typhoon Trio (GE Healthcare Life Sciences).

BsSMC stock solution was supplemented with a 1.1-fold excess of Cy3 NHS ester (GE Healthcare) dissolved in dimethylsulphoxide and left overnight at 4 °C. Free dye was removed using Micro Bio-Spin P-30 Gel Columns (Bio-Rad). We found that this step was crucial, since using a centrifugal concentrator to remove free dye abolished the protein's activity. After running the sample on an SDS–PAGE gel, the labelling was confirmed using a Typhoon imager (GE Healthcare), and the labelling efficiency was calculated by measuring absorbance at both 280 and 550 nm using a NanoDrop spectrophotometer (Thermo Scientific). The labelling efficiency was around 50% (approximately one Cy3 dye per BsSMC dimer).
The compaction rate with unlabelled WT BsSMC (Fig. 4a) was used to gauge activity of the labelled WT BsSMC. When a high concentration of Cy3-labelled BsSMC was flowed in, a spectrally distinct quantum dot attached at the end of the flow-stretched DNA was imaged onto the other half of an electron multiplying charge coupled device (EMCCD) camera. The position of the quantum dot was determined by Gaussian fitting and used to determine the compaction rate. The rate of compaction by labelled BsSMC was comparable to that by unlabelled BsSMC, indicating that labelling does not disrupt the DNA-compacting activity of the protein.