Peripheral venous blood samples were collected in EDTA tubes, and PBMCs were isolated within the first 4 hours after extraction, using a Ficoll-Paque gradient method.  Total RNA including miRNAs were isolated from PBMCs with the miRNeasy Mini kit (Qiagen).  Quality and integrity of the RNA were evaluated by the Bioanalyzer 2100 with Agilent RNA 6000 Nano kit (Agilent). Only those samples with RIN > 7.5 were sequenced. Small RNA libraries were constructed with Illumina's TruSeq Small RNA kit v.4 (Illumina, ref. RS-200-0012). In brief, 1 microgram of total RNA was used, 3′ and 5′ adapters were sequentially ligated to the ends of RNA < 200 nt long, and reverse transcribed to generate cDNA (SuperScript II, ref. 18064-014, Invitrogen) with a specific primer (RNA RT Primer) complementary to the 3' RNA adapter. The cDNA was further amplified by PCR (11 cycles) using a common primer complementary to the 3′ adapter, and a 5′ primer containing 1 of 48 index sequences. Libraries were size-selected using 6% Novex® TBE Gels (ref. EC6265BOX, Life Technologies). Fragments with insert sizes of 18 to 36 bp were cut from the gel, and DNA was purified, quantified and pooled for multiplexed sequencing. Final libraries were analyzed using Agilent DNA 1000 chip to estimate the quantity and check size distribution, and were then quantified by qPCR using the KAPA Library Quantification Kit (ref. KK4835, KapaBiosystems) prior to amplification with Illumina's cBot.
Experimental Factor: clinical history
HIV-1 coinfection with hepatitis C virus