Illumina HiSeq 3000 sequencing; RNA-Seq of SOX4 and HOXD3 CRISPR KO SupT1 cells against wildtype SupT1 cells during HIV-1 infection
RNA-Seq of SOX4 and HOXD3 CRISPR KO SupT1 cells against wildtype SupT1 cells during HIV-1 infection
RNA from at least 1x10^6 cells from uninfected and cells 8, 18, 30 and 42h post infection was isolated with Qiazol (Qiagen, #79306). Cells were cultured in RPMI 1640 ATCC formulation (Thermo Fisher Scientific, #A1049101) supplemented with 10% fetal bovine serum (FCS) (Thermo Fisher Scientific, #10270106), additional 0.68 nmol/ml L-glutamine (Sigma-Aldrich, #G7513-100ml), and Penicillin-Streptomycin (PenStep,\#P0781) to final 100 U/ml Penicillin and 100 µg/ml Streptomycin (Sigma-Aldrich, #P0781), and incubated at 37°C and 5% CO2. Sup-T1 cells were maintained as suspension cultures of 2x10^5 -2x10^6 cells/ml. NLENG1-IRES G1I is a full-length CXCR4-tropic molecular clone of HIV derived from pNL4-3 and carries enhanced green fluorescent protein (eGFP) inserted in front of an IRES sequence and Nef. The plasmid was a kind gift from Dr. David N. Levy, New York University College of Dentistry.1200ng p24 HIV-1 eGFP was added to 1x10^6 Sup-T1 cells in 1 ml RPMI 1640 ATCC formulation supplemented with 10% FCS, PenStrep and 10 µg/ml polybrene (Santa Cruz Biotechnology, #sc-134220). To facilitate virus attachment, spinofection was carried out by centrifugation at 1200 g for 2 hours at 20°C. The supernatant was aspirated, and the cells were cultured in complete media for the indicated time points. base calls provided by the Illumina Real-Time Analysis (RTA) software were subsequently converted into BAM format (Illumina2bam) before de-multiplexing (BamIndexDecoder) into individual, sample-specific BAM files via Illumina2bam tools (1.17.3).  BAM files were converted to fastq files with BEDTools (2.27.1) and quality assessed with FastQC (0.11.7). RNA was isolated with Qiazol (Qiagen, #79306) and purified and DNAse treated on Direct-zol columns (Zymo Research, #R2052). RNA integrity was controlled using the Caliper LabChip GX (Perkin Elmer), RNA Quality Scores were found to be 10-8.16. RNA-seq libraries were prepared with the TruSeq Stranded mRNA LT sample preparation kit (Illumina) using both, Sciclone and Zephyr liquid handling robotics (PerkinElmer). Library concentrations were quantified with the Qubit Fluorometric Quantitation system (Life Technologies) and the size distribution was assessed using the Experion Automated Electrophoresis System (Bio-Rad).