Precast gel

Protein extracts were obtained as in [2 (link), 12 (link), 40 (link)]. Specifically, samples were lysed in buffer containing 50mM Tris-HCl (pH 7.4), 250 mM NaCl, 0.1% tritonX100, 5mM EDTA, 0.3% Empigen BB and supplemented with 1mM PMSF and protease inhibitor mix. Western Blot assay was performed using 40 μg of total extract. Co-immunoprecipitation experiments were performed using 4 μg of antibody for a range of 500-700 μg of protein extract. Ademtech's Bio-Adembeads paramagnetic bead system was used to immunoprecipitate the specific proteins. A negative control was performed with the same amount of protein extract sample immunoprecipitated with the corresponding purified IgG (Santa Cruz). Total proteins were resolved by SDS-PAGE using a 3-8% gradient and/or 10% Invitrogen Precast gel (NuPage, TA or MES buffer). Specific protein signals were revealed with ECL Prime (Amersham, GE Healthcare) and detected by UVIDOC (Eppendorf S.r.l.). The intensity of each band was evaluated by using UVIDOC and/or the NIH Image J 1.8 software (National Institutes of Health, Bethesda, Maryland, USA). Optical density values of specific proteins were normalized to that of tubulin, H1, HSP90 or fibrillarin.

Cell lysates were separated in 4–12% pre-cast gel (Invitrogen) and transferred to PVDF membranes. Membranes were blocked with 5% FBS and primary antibodies were applied at 4 °C overnight or at RT for 2 h. After washing, membranes were incubated with HRP-conjugated secondary antibodies for 20 min (Pierce Fast Western Blotting Blot Kit) at room temperature. Finally, blots were washed three times and visualized using ECL substrate (Pierce Fast Western Blotting Blot Kit). Antibodies and dilutions were used as follows: anti-PCIF1 antibody at 1:1000 (Proteintech, 16082-1), anti-p24 antibody at 1:1000 (Abcam, ab9071), anti-p62 antibody at 1:1000 (Abcam, ab56416), anti-Vpr antibody at 1:1000 (Proteintech, 51143-1-AP), anti-Vpu antibody at 1:1000 (NIH AIDS Reagent Program, Cat# 969), anti-Nef antibody at 1:1000 (NIH AIDS Reagent Program, Cat# 1539), anti-Vif antibody at 1:1000 (NIH AIDS Reagent Program, Cat# 6460), anti GAPDH-HRP antibody at 1:5000 (Proteintech, HRP-60004), mouse anti-FLAG M2 antibody at 1:1000 (Sigma-Aldrich, F1804), anti-HDAC1 antibody at 1:1000 (Abcam, ab7028), anti ETS1 antibody at 1:2000 (Proteintech, 12118-1-AP), anti-GFP-tag antibody at 1:1000 (Proteintech, 50430-2-AP), HA antibody at 1:1000 (Santa-Cruz, sc-805). The density of western bands were quantified by ImageJ (https://imagej.nih.gov/ij/). GAPDH was used as a loading control.

Small RNA libraries were prepared using the NebNext Small RNA Library Preparation Kit (New England Biolabs) for miRNA and short non-coding RNAs sequencing according to manufacturer's instructions, starting from 200 ng RNA and using 15 PCR cycles. Each library was analyzed with the High Sensitivity DNA chip (Agilent) using Agilent 2100 Bioanalyzer. Pools of 12 libraries were concentrated with AMPure XP magnetic beads (Beckman Coulter) and eluted in 40 μl of nuclease free water. The eluate from each pool was loaded onto a 6% precast gel (Life Technologies), a ≈146 bp band was cutted, purified with a 5 μl filter with Qiaquick gel extraction kit (Qiagen) and quantified using the Qubit DNA HS (Life Technologies). The miRNAseq libraries were sequenced on a NextSeq500 sequencer (Illumina) using 75 bp-reads and producing on average 2,047,034,738 reads per sample.

Small RNA libraries were prepared using Small RNA Library Preparation Kit NEBNext (New England Biolabs) according to the manufacturer's instructions starting from 40 ng of EV-derived RNA and using 15 PCR cycles. Each library was analyzed with the High Sensitivity DNA chip (Agilent) using Agilent 2100 Bioanalyzer. Pools of 12 libraries were concentrated with AMPure XP magnetic beads (Beckman Coulter) and eluted in 40 μL of nuclease free water.
The eluate from each pool was loaded onto a 6% precast gel (Life Technologies), and a ≈146 bp band was cut and put in 300 μL of nuclease free water overnight in 2 mL low bind Eppendorf tube. Gel debris was removed with QIAquick Gel Extraction Kit (Qiagen), and cDNA was precipitated using 3M NaOAc, 100% cold ethanol and glycogen, centrifuged at 20,000 g for 20 min at 4°C. After one wash with 70% ethanol, cDNA was resuspended in 10 μL nuclease free water and quantified using the Qubit DNA HS (Life Technologies). Pool was analyzed with the High Sensitivity DNA chip (Agilent) using Agilent 2100 Bioanalyzer. Two pools (2 × 12 samples purified indexed libraries) were run at the concentration of 1.6 pM on the NextSeq500 sequencer (Illumina) according to the manufacturer's instructions, in 75 nts single-end sequencing mode.

Immunoblotting was performed as previously described^{55 (link)}. Briefly, whole cell extracts were obtained by incubating the cells in lysis buffer supplemented with protease and phosphatase inhibitor cocktail. Lysates concentration was determined and equal amounts of proteins were loaded on a 4–12% precast gel (Thermo Fisher Scientific) and transferred to nitrocellulose membranes. Blots were blocked with TBST 5% non-fat dry milk (Bio-Rad Laboratories, Hercules, CA, USA) and incubated overnight at 4 °C with primary antibodies (described in the Antibodies and Reagents section) then incubated for 45 min with secondary HRP-conjugated antibodies dissolved in TBST/1% BSA. Chemiluminescent signals were detected with Amersham ECL prime or select western blotting detection reagent (GE Healthcare Life Sciences, Barrington, IL, USA). Images were taken and analyzed with Bio-Rad ChemiDoc Imagers (Bio-Rad Laboratories).