Illustra microspin s 400 hr column

Molecular cloning and determination of the nucleotide sequences of HIV-1 strains passaged in the presence of each compound were performed as previously described (Aoki et al., 2009 (link)). In brief, high-molecular-weight DNA was extracted from HIV-1-infected MT-4 cells by using the InstaGene Matrix (Bio-Rad Laboratories, Hercules, CA) and was subjected to molecular cloning, followed by nucleotide sequence determination. The PCR primers used for the protease-encoding region were KAPA-1 (5’-GCAGGGCCCCTAGGAAAAAGGGCTGTTGG-3’) and Ksma2.1 (5’-CCATCCCGGGCTTTAATTTTACTGGTAC-3’). The PCR mixture consisted of 1 μl proviral DNA solution, 10 μl Premix Taq (Ex Taq Version; Takara Bio Inc., Otsu, Japan), and 10 pmol of each PCR primer in a total volume of 20 μl. The PCR conditions used were an initial 1 min at 95°C, followed by 30 cycles of 30 s at 95°C, 20 s at 55°C, and 1 min at 72°C, with a final 7 min of extension at 72°C. The PCR products were purified using spin columns (illustra MicroSpin S-400 HR columns; GE Healthcare Life Science., Pittsburgh, PA), cloned directly, and subjected to sequencing using a model 3130 automated DNA sequencer (Applied Biosystems, Foster City, CA).

The methods of ribosome profiling and sequencing were consistent with those used in previous experiments successfully performed on tea plants^{30 (link)}. Briefly, three replicate samples from the CK and LT treatments were ground in liquid nitrogen and dissolved in a lysis buffer. The lysates were centrifuged, and the supernatants were collected for RF preparation. Five microliters of DNase I and 7.5 µL of RNase I were added to 300 µL of lysate and incubated for 45 min, and the reaction was stopped by adding SUPERase·In RNase inhibitor. Subsequently, the size exclusion column (Illustra MicroSpin S-400 HR Columns, GE Healthcare) was equilibrated with 3 mL of polysome buffer by gravity flow and centrifuged. RFs larger than 17 nt were isolated by an RNA Clean and Concentrator-25 kit (Zymo Research, Beijing, China). After removing the rRNA, the RFs were purified, and the library was constructed. Sequencing was performed by Gene Denovo Biotechnology (Guangzhou, China) on an Illumina HiSeqTM 2500.

Community DNA was extracted from 0.5 g aliquots of each soil sample using a PowerSoil DNA extraction kit (Mo Bio Laboratories, Inc., Carlsbad, CA, USA). Extracted DNA was purified with illustra MicroSpin S-400 HR columns (GE Healthcare Bio-Sciences Corp, Piscataway, NJ, USA) and quantified using a Quant-iT PicoGreen dsDNA assay kit (Invitrogen Corp, Carlsbad, CA, USA).

The 7-day-old Col-0 and vir-1 seedlings were subjected to 0 or 4 h of high light treatment, immediately frozen in liquid nitrogen for at least 1 h, ground into a fine powder in liquid nitrogen, and dissolved in 400 µl of lysis buffer (20 mM Tris·HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 100 µg/ml cycloheximide, and 1% [v/v] Triton X-100). Following incubation on ice for 10 min, the lysate was centrifuged at 17,000 g for 10 min at 4 °C. To prepare ribosome footprints (RFs), 10 µl of RNase I and 6 µl of DNase I were added to 400 µl clarified lysate. After incubation for 45 min at room temperature, 10 µl of SUPERase-In RNase inhibitor was added to stop the nuclease digestion. The digested lysate was placed into a pre-equilibrated size exclusion column (Illustra MicroSpin S-400 HR Columns; GE Healthcare) and eluted from the column by centrifugation at 600 g for 2 min. An RNA Clean and Concentrator-25 kit (Zymo Research) was used to isolate RFs larger than 17 nt. A probe was used to remove rRNA, and the RFs were further purified with magnetic beads (Vazyme)^{89 (link)}. NEBNext Multiple Small RNA Library Prep Set for Illumina (NEB) was used to construct the Ribo-seq libraries. Sequencing was performed on an Illumina HiSeq 2500 platform by Gene Denovo Biotechnology Co. (Guangzhou, China).

Ribosome profiling was conducted using a method described in a previous report [32 (link)]. Briefly, 50 mL E. coli culture was collected after 5 min of treatment with chloramphenicol (34 mg/mL) at an exponential growth phase. Cells were flash frozen with 0.5 mL lysis buffer (1% Triton X-100, 34 μg/mL chloramphenicol, 133 mM NaCl, 4.75 mM MgCl₂, and 19 mM Tris-HCl, pH 7.5) and lysed using a mortar and pestle. Then, the supernatant containing 100 μg RNA was treated with 2,000 gel units of micrococcal nuclease (MNase; NEB). Polysomes were recovered from MNase-digested samples using Illustra MicroSpin S-400 HR columns (GE Healthcare, Chicago, IL, USA) followed by phenol:chloroform:isoamyl alcohol extraction. rRNA was removed from 1 μg polysome-protected RNA using a Ribo-Zero rRNA Removal Kit according to the manufacturer’s instructions. The rRNA-subtracted RNA samples were phosphorylated by treating 10 U T4 polynucleotide kinase (NEB) at 37°C for 1 h and purified with RNeasy MinElute columns (Qiagen, Hilden, Germany). Sequencing libraries were prepared from phosphorylated RNA samples using the NEBNext Small RNA Library Prep Set for Illumina (NEB) according to the manufacturer’s instructions. The library was sequenced using an Illumina platform. Information on NGS (run recipe and instrument) is summarized in S6 Table.

Recombinant Human SDF and the SDF ELISA kits used were obtained from R&D Systems (Abingdon, UK). Illustra MicroSpin S-400 HR columns were purchased from GE Healthcare (Amersham, UK). Float-A-Lyzers were obtained from Spectrum Labs (Amsterdam, The Netherlands). EndoGrow cell culture medium was purchased from Merck Millipore Ltd. (Cork, Ireland). Growth factor reduced Matrigel^® was purchased from Corning BV (Amsterdam, The Netherlands). Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza Ltd. (Slough, UK). All other chemicals and reagents were sourced from Sigma Aldrich (Dublin, Ireland).
L-PGA and Star-PGA were synthesized by Dr Robert Murphy using the method previously outlined by Byrne et al. [38 (link)]. The L-PGA, shown in Figure 1a contained 200 glutamic acid residues per molecule (Mn: 26 kDa). The star-PGA, shown in Figure 1b, had a polypropyleneimine (PPI) core and eight arms, each with 40 glutamic acid residues (theoretical Mn: 42 kDa; estimated isoelectric point: 4.1). Gel permeation chromatography was used to ensure polymer formation. The chromatograms obtained were analogous to those previously reported by our group [47 (link)].

Sample preparation for ribosome profiling was conducted according to the manufacturer’s specifications for the TruSeq Ribo Profile (Mammalian) Library Prep Kit (Illumina). Briefly, HEK293 cells were treated with CHX (Sigma-Aldrich, final concentration 0.1 mg/ml) for 1 min. In-dish cell lysis was performed using mammalian lysis buffer (including CHX at a concentration of 0.1 mg/ml). Then 600 μl of lysate were taken and 15 μl of RNase I (100 U/μl, Thermo Fisher Scientific) were added and the mixtures were incubated for 45 min at room temperature, followed by adding 15 μl SUPERaseIn RNase Inhibitor (Ambion, Thermo Fisher Scientific) to stop the reaction. Ribosome recovery was performed by illustra MicroSpin S-400 HR Columns (GE Healthcare) and the RPFs were purified by RNA Clean & Concentrator (Zymo Research). Ribosomal RNAs were depleted using Ribo-Zero Magnetic Gold Kit (Human/Mouse/Rat, Illumina). RPFs without ribosomal RNA were run on a 15% urea denaturing-PAGE gel, and the gel slices corresponding to 28–30 nts were excised. The RPF RNAs were eluted and precipitated followed by library construction according to the manufacturer’s protocol.