Rna 6000 pico

RNA was isolated from each enriched cell population specimen using miRNease Micro kits from Qiagen (Germantown, MD) following the manufacturer’s instructions, with modification. RNA was eluted 3 times, each time with 17 μL RNase-free H₂O, and all the eluents were combined. DNase treatment was performed using a Turbo DNA-free Kit from Thermo Fisher Scientific. After the DNase treatment, RNA was concentrated using an RNA Clean & Concentrator kit from Zymo Research (Irvine, CA). Quantitative assessment of the total RNA yield was performed using the Qubit Broad Range RNA kit from Thermo Fisher Scientific. Before RNA-Seq analysis, the integrity of the RNA was evaluated using a 2100 Bioanalyzer and RNA 6000 Pico kits from Agilent Technologies (Santa Clara, CA). RNA samples accepted for RNA-Seq analysis had RIN values in the range of 6.3–8.

Total RNA from whole blood, collected in Tempus tubes, was isolated with a Tempus™ Spin RNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA) and treated with DNase using a TURBO DNA-free™ Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocol. RNA purity and concentration were investigated via a NanoDrop ND-1000 spectral photometer (Thermo Fisher Scientific, Waltham, MA, USA). The integrity of RNA was determined with a 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) using RNA 6000 Pico or Nano LabChip Kits (Agilent Technologies, Waldbronn, Germany) according to the manufacturer’s instructions. RIN values of investigated samples were in the range of 7.6–9.9 and no ribosomal 18S or 28S peaks were detected in the profiles.
RNA sequencing libraries were generated with the Illumina TruSeq^® Stranded mRNA, including the Ribo-Zero Globin technology that depletes cytoplasmic and mitochondrial rRNA and globin mRNA. RNA sequencing was performed by IMGM Laboratories GmbH (Martinsried, Germany) on the Illumina NextSeq^® 500 next-generation sequencing system with 1 × 75 bp single-read chemistry. Raw files are accessible under the Gene Expression Omnibus accession number GSE174825.

Total RNA was isolated from the ex vivo single-dose simulation study cell pellets (described above) using the RNeasy minikit (Qiagen, Valencia, CA) according to the manufacturer’s protocol with the following modifications. Samples were homogenized using 600 μl RLT lysis buffer (supplied with kit) plus β-mercaptoethanol and 5 mm stainless steel beads in a TissueLyser instrument (Qiagen, Valencia, CA); RNA was eluted with 30 μl RNase-free water. RNA samples were quantitated, and the RNA integrity number (RIN) was determined using the RNA 6000 Nano (Jurkat and HCT116) or RNA 6000 Pico (CD4) kits and the 2100 Bioanalyzer instrument (Agilent, Santa Clara, CA) according to the manufacturer’s protocol. RNA (1 μg) for each sample was sent to Quintiles-Quest Expression Analysis, Inc. (Morrisville, NC) for RNA-Seq analysis. RNA samples were converted into cDNA libraries using the Illumina TruSeq stranded total RNA sample preparation kit (catalog no. RS-122-2303; Illumina). Final libraries were quantified via qPCR, normalized to 2 nM, pooled, and sequenced on the Illumina platform. Sequences from the RNA-Seq analysis were analyzed using DEseq2 software to evaluate genes modulated with fragments per kilobase per million (FPKM) of >50 and a false-discovery rate (FDR) of <0.1 for each time point.

Extracted and DNase-treated RNA was quantified using the Qubit 4 Fluorometer (ThermoFisher) with the High Sensitivity RNA reagents and Bioanalyzer (Agilent) with RNA 6000 Pico reagents. Ribosomal depletion, DNA conversion, and library preparation was performed on all samples using the Illumina TruSeq Stranded Total RNA kit. 151 base pair reads were sequenced on the Illumina NextSeq. Across fifteen samples (three independent experiments x five time points) the total number of reads generated for each sample ranged from approximately 26 million to 40 million reads. Sequencing data was quality trimmed using FaQCs [56 (link)] with a quality score cutoff of Q20. Differential expression analysis was performed using PiReT [57 ] V 0.3.2 and utilizing DEseq2 [58 (link)] default parameters and setting a q-value of 0.05 (false discovery rate metric). The experimental design file (provided in the supplementary material) was used to dictate the replicate sample ID’s and sequencing data to be used in the PiReT analysis. Human genome version hg38 was used as the reference genome. KEGG [59 (link), 60 (link)] pathway mapping was performed using Omics Pathway Viewer - ‘OPaver’ (Li, unpublished). Raw RNA-Seq reads were deposited in the NCBI SRA database under the accession numbers SRR11994167- SRR11994181. Metadata for each sample are also accessible under NCBI BioProject PRJNA638768.

RNA was purified from whole-blood cultures using MagMax for Stabilized Blood Tubes RNA Isolation Kit (Thermo Fisher Scientific). Manufacturer’s instructions were followed, but the initial homogenization volumes were proportionally adjusted to the sample input volume. RNA from cell suspensions was purified using the RNAqueous-Micro Total RNA Isolation Kit (Thermo Fisher Scientific). RNA integrity was assessed using RNA 6000 Pico or RNA 6000 Nano assay on Agilent 2100 Bioanalyzer (Agilent Technologies). RNA concentration and purity were assessed using the NanoDrop 8000 (Thermo Fisher Scientific). All procedures were performed according to the manufacturer’s instructions.