Rnazol rt

Total messenger RNA from zebrafish embryos aged at 3 dpf were extracted by the RNAZol^®RT (Invitrogen), and quantified with NanoDrop (Thermo Scientific, Waltham, MA, USA). RevertAid first cDNA synthesis kit (K1622, Fermentas, Waltham, MA, USA) was used to synthesize first-strand cDNA from total zebrafish RNA according to the manufacturer’s instructions. RT-qPCR was performed using iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) on a Bio-Rad iCycler using β-actin as control, and data were analyzed using the ∆∆Ct method [39 (link)]. The primer sequences used to perform quantitative real-time PCR are listed in Table 1.

Total RNA was extracted from cCFU-Fs or isolated cCFU-F exosomes by RNAzol RT (Invitrogen) following the manufacturer’s instructions. The RNA concentrations were determined by using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). Isolated RNAs were polyadenylated using an NCode miRNA First-Strand cDNA Synthesis Kit (Invitrogen). qRT-PCR was performed with SYBR Green I (DH Biotech, Shanghai) and Prism 7500 SDS (Applied Biosystems; Thermo Fisher Scientific, Inc.). Amplification was performed at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. For mature miRNA expression, the universal primer provided in the NCodeTM miRNA First-Strand cDNA Synthesis Kit was used with one of the following forward primers:
Mmu-miR221: 5′-GUCAACAUCAGUCUGAUAAGCUA-3′and Mmu-miRU6: 5′-ACACGCAAATTCGTGAAGC-3′.
The relative gene expression values were calculated using the ΔΔCt method (ΔΔCt = ΔΔCt treated- ΔΔCt untreated control) with the equation y = 2-ΔΔCt, and U6 was used as a control.

DNA was extracted from CD4⁺ T cell subsets sorted from peripheral blood and gut samples collected from 6 participants who were on ART for at least 15 years (Table 1). Four hundred microliters of RNAzol RT (MRC, Inc.) was added to a 1.5-ml Eppendorf tube containing the cell pellet; a 0.4× RNAzol RT volume of sterile nuclease-free water (Invitrogen) was added and mixed by inversion for 15 s, followed by incubation for 15 min. The mixture was centrifuged at 16,000 × g for 15 min at room temperature. The top phase was removed, and the bottom phase was used for DNA extraction. Nine hundred microliters of DNAzol (MRC, Inc.), followed by 10 μl of glycogen (20 μg/μl; Qiagen), was added to the bottom phase. DNA was precipitated by adding 500 μl of 200 proof ethanol (Sigma-Aldrich). The mixture was incubated for 10 min at room temperature and centrifuged at full speed for 30 min. The supernatant was removed, and the DNA pellet was washed with 75% ethanol twice. The pellet was air dried until no ethanol was visible. The pellet was dissolved in 300 μl of 8 mM NaOH (Sigma-Aldrich), followed by neutralization by adding 24 μl of 0.1 M HEPES (Gibco).