Globinclear

This laboratory study aimed to test whether and how deep sequencing of TCR repertoires from HIV patients might produce results of interest to clinical and immunological investigations. Adult HIV⁺ ART-naïve patients due to start treatment were recruited, with the first sixteen eligible candidates being processed. Exclusion criteria included febrile or AIDS-related illness; tumors; coinfection with Hepatitis B or C; immunomodulatory therapy, recent vaccination, or breaks in treatment. 2.5 ml of peripheral blood was drawn into Tempus tubes, and RNA was extracted as per the manufacturer’s instructions (Life Technologies). Residual gDNA was removed using the TURBO-DNase kit, and globin mRNA was depleted using GLOBINclear (Life Technologies). RNA was likewise prepared from consenting adult healthy controls. Human participation in this study was approved by UK Research Ethics Committee. All subjects gave written informed consent in accordance with the Declaration of Helsinki.

5 ml of healthy adult volunteer blood was drawn into Tempus Blood RNA tubes (Life Technologies) and RNA was extracted using the Tempus RNA isolation kit (Life Technologies). Residual DNA was removed using the TURBO DNase kit, and globin mRNA was depleted using GLOBINclear (both Life Technologies).
The KT2 T cell clone was a gift of Prof. A. Lanzavecchia (Institute for Research in Biomedicine, Bellinzona, Switzerland). The clone was grown as described27 (link). RNA was isolated using the RNeasy Mini Kit (Qiagen). RNA was treated with RQ1 DNase (Promega) following manufacturer’s instructions to remove any residual genomic DNA.
Two different protocols were used to amplify and then sequence the T cell receptor chains. All primers are from Sigma-Aldrich and sequences can be found in Table 1.

Groups of five C57 mice were vaccinated with BCG or treated with PBS as described above. Cardiac blood was collected at 14 days after vaccination, and RNA was purified. RNA derived from blood was treated with GlobinClear (ThermoFisher Scientific) to deplete globin transcripts (27). All RNA samples were treated with DNase to remove contaminating DNA, and then purified using QiaQuick Spin kits (Qiagen). Purified RNA was quantified using a Nanodrop (ThermoFisher Scientific), and the quality of extracted RNA was verified by Bioanalyzer 2100 (Agilent); all samples had an RNA Integrity Number (RIN) greater than 8.5. RNA prepared from blood from five mice was pooled by group within individual experiments, and then pooled RNA samples from three independent experiments using naïve and BCG-vaccinated animals were subjected to RNASeq analyses. Amplification and labeling of RNA samples were performed by the CBER/FDA Sequencing Core Facility using Illumina TotalPrep RNA Amplification (Applied Biosystems) and using an input of 1 μg of total RNA per sample. Sequencing was performed on a MiSeq PE250 platform (Illumina). The RNASeq data have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number (GSE212586).

Whole blood was collected from IMRAS trial participants directly into PAXgene blood RNA tubes (PreAnalytiX, Hombrechtikon, Switzerland) and stored at −20°C. RNA extraction and globin transcript depletion (GlobinClear, ThermoFisher Scientific, MA, USA) were performed prior to cDNA library preparation using the Illumina TruSeq Stranded mRNA sample preparation kit (Illumina, CA, USA). Globin transcript depletion, cDNA library preparation and RNA sequencing were performed by Beijing Genomics Institute (Shenzhen, China). A total of sixty-six RNA-seq samples were sequenced, with a target depth of 30 million reads per sample. Eleven of the samples were sequenced on Illumina (San Diego, CA) Hiseq2000 sequencers using 75 base-pair (bp) paired-end reads. The remaining one hundred and eighty-six samples were sequenced on BGI500 sequencers using 100 bp paired-end reads.

In addition to the subset of individuals with detailed separate cell samples (n=60), a further eight patients were included in gene expression array analyses (total n=68). Separated cell RNA was extracted using the Allprep DNA/RNA miRNA universal kit (Qiagen). Whole blood RNA was extracted from PAXgene tubes using the PAXgene blood miRNA kit (PreAnalytix, Switzerland). The RNA was quantified and assessed for quality using the Agilent BioAnalyzer with only samples with a RNA-integrity number of >7 being used for downstream analyses. Following sample concentration and cleanup using the MinElute RNA cleanup kit (Qiagen), globin mRNA transcripts were depleted using GlobinClear (Ambion, Life Technologies USA). RNA was amplified and biotylated using the Illumina TotalPrep RNA Amplification Kit (Ambion, Life Technology). The cRNA was quantified and assessed for quality using the Agilent BioAnalyzer with the expected gel appearance of cRNA is a ‘smear', with a distribution of cRNA size is expected between 250–5,500 nucleotides, with most cRNA between 1,000 and 1,500 nt. Illumina HT12 human v4 expression microarrays were performed using a hybridization time of 18 h at 58 °C. Data were analysed using the lumi55 (link) and limma61 packages. Data were background adjusted, variance stabilized and quantile normalized.