title: Sample 5
organism: Homo sapiens
description: Protocols: Human samples, cell isolation and TCR-stimulation Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats of healthy individuals by Ficcol-Paque PLUS (GE Healthcare, Uppsala, Sweden) density gradient centrifugation. Subsequently, naïve CD4+ T cells were magnetically isolated by negative selection using the EasySep™ Human Naïve CD4+ T Cell Enrichment kit (Stem Cell Technologies), according to the manufacturer's instructions, with purity was always above 97% as assessed by flow cytometry (CD45RA+CD45ROnegCD4+CD3+ cells). Isolated naive cells were cultured in complete culture medium: RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 2mM L-glutamine, 100 U/ml penicillin -100μg/ml streptomicin, and 0,05mg/ml Gentamicin (all from Invitrogen). For TCR-stimulation, naïve CD4+ T cells were cultured for 72h at 2,5x106 cells/ml in complete culture medium in the presence of plate-bound anti-CD3 (1μg/ml, clone OKT3, eBioscience), soluble anti-CD28 (1μ/ml, clone 28.2, BD-Pharmingen) and 10IU/ml of IL-2 (Dr. Maurice Gately, Hoffmann-La Roche Inc, NIH AIDS Reagent Program). For prolongued time-course assays, cells were switched to medium supplemented with IL-2 after the initial 72h culture. Virus production and HIV infections 293T cells were transfected with pRod (NIBSC - Centre for AIDS Reagents, Medical Research Council UK) or pNL4-3 (NIH AIDS Reagent Program) for 24 hours using fuGENE HD Transfection Reagent (Roche), according to the manufacturer's instructions. Cell-free culture supernatants were quantified in terms of reverse transcriptase (RT) activity using a Cavidi Lenti RT Activity Kit (Cavidi Tech AB, Uppsala, Sweden), according to manufacturer's instructions. Viral stocks were concentrated by ultracentrifugation (30 minutes at 50,000g and 4ºC using a Beckmann L8-60M ultracentrifuge) before being resuspended in complete medium supplemented with polybrene (3µg/ml; Sigma) and used to infect primary cells or stably-transfected cell lines, at a dose of 0.853ng RT/106 cells. After an initial culture period of 3 hours under standard conditions (37ºC/5%CO2) cells were washed to remove residual virus. Infected unstimulated and TCR-stimulated naïve CD4+ T cells were resuspended in medium supplemented with 10IU/ml IL-2 and polybrene (3µg/ml), and cultured at 37ºC for a total of 24 hours. After this period, cells and supernatants were collected and stored immediately at -80ºC. HIV-1NL43 infected cell lines (JØ and J34c, see below) were cultured in complete medium supplemented with polybrene (3µg/ml) and 1µg/ml G418 (Life technologies) for a total of 48 hours. Culture supernatants and cell pellets were harvested at 24 and 48 hours. Infection was assessed by quantifying cell-associated viral DNA and gag and tat mRNA levels were quantified as described previously (Soares et al., 2011). Viral production was assessed by measuring RT activity in supernatants using SYBR green product-enhanced RT (SG-PERT) as previously described (Nunes-Cabaço et al., 2015). Total RNA from naïve CD4+ T cells was isolated using mirVana miRNA Isolation kit (Ambion) according to the manufacturer's instructions. Trizol reagent (Ambion) was used for isolation of total RNA from Jurkat cells, according to the manufacturer's instructions. RNA quantification was performed on a ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). For small-RNA-seq and microarray analysis, the quality of total RNA was validated using a 2100 Bioanalyzer (Agilent Technologies), RNA 6000 Nano LabChip kits, and 2100 expert software for individual samples. Only samples without visible degradation in the Bioanalyzer profile were selected for further downstream analysis. Total RNA samples from multiple individuals were pooled for the generation of sequencing libraries (see below) and re-quantified using Qubit (Life technologies). Small RNA-Seq design and sequencing The small-RNA-seq libraries used in this study were generated in three distinct experiments by outsourcing. Total RNA samples were gel fractionated and fragments between 15 and 30bp were purified and used to generate small RNA-seq libraries according to Illumina's Small RNA-seq protocol. Dataset 1 was generated in a pilot experiment in which total RNA samples were generated for each condition (naïve and activated CD4+ T-cells) by pooling equal amounts of total RNA obtained from 9 individuals to generate a single RNA pool per condition. Small-RNA-seq libraries derived from these samples were sequenced using the Genome Analyser II sequencer (Illumina), in separate lanes, occupying the whole flow cell using the Chrysalis 36 cycles v 4.0 sequencing kit. Dataset 2 was generated in a second experiment, in which equal amounts of total RNA obtained from three individuals were pooled in balanced sets of three, generating to two replicate RNA pools per condition (quiescent naïve, quiescent naïve infected with HIV-1 and quiescent naïve infected with HIV-2), from a total of six donnor. Dataset 3 was generated in a third experiment in which total RNA samples from 3 individuals were pooled per condition (stimulated naïve, stimulated naïve infected with HIV-1 and stimulated naïve infected with HIV-2) and three replicate libraries were generated from a total of 9 donnors. Libraries from the second and third experiments were sequenced using the HiSeq2000 sequencer (Illumina) using the TruSeq(TM) SBS v5 sequencing kit, occupying 20% of the lane. The same 3' sequencing adaptor (ATCTCGTATGCCGTCTTCTGCTTGT) was used in all experiments allowing comparison of results and was removed from raw data before analysis. Raw sequencing data from dataset 1 was pre-processed using the SCS.2.6 and CASAVA.1.6 pipeline (Illumina). Raw reads from datasets 2 and 3 were pre-processed using the HiSeq Control Soft. V 1.3.8, RTA 1.10.36, CASAVA 1.7.0 and OLB-1.9.0 (Illumina).
attributes.tag: ENA first public
attributes.value: 2016-12-21
attributes.tag: ENA last update
attributes.value: 2016-12-13
attributes.tag: External Id
attributes.value: SAMEA26576668
attributes.tag: INSDC center alias
attributes.value: BioISI - Biosystems, Integrative Sciences Institute Instituto de Medicina Molecular University of Lisbon
attributes.tag: INSDC center name
attributes.value: BioISI - Biosystems, Integrative Sciences Institute Instituto de Medicina Molecular University of Lisbon
attributes.tag: INSDC first public
attributes.value: 2016-12-21T17:01:34Z
attributes.tag: INSDC last update
attributes.value: 2016-12-13T14:31:02Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-4214:Sample 5
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: CD4-positive helper T cell
attributes.tag: common name
attributes.value: human
attributes.tag: phenotype
attributes.value: stimulated-naive T cell CD4+
attributes.tag: sample name
attributes.value: E-MTAB-4214:Sample 5