Sciclone ngs workstation

Total RNA was isolated using the AllPrep DNA/RNA Mini Kit (Qiagen). RNA amount was measured using Qubit 2.0 Fluorometric Quantitation (Life Technologies), and the RNA integrity number was determined using Experion Automated Electrophoresis System (Bio-Rad). RNA-seq libraries were prepared using a Sciclone NGS Workstation (PerkinElmer) and a Zepyhr NGS Workstation (PerkinElmer) with the TruSeq Stranded mRNA LT sample preparation kit (Illumina). Library amount and quality were determined using Qubit 2.0 Fluorometric Quantitation (Life Technologies) and Experion Automated Electrophoresis System (Bio-Rad). The libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq 3000/4000 platform and the 50-bp single-read configuration.

Three biological replicates from each genotype were prepared for RNA-sequencing. Total RNA of in vitro generated CTLs (6 days after activation) was isolated with RNeasy kit (Qiagen) combined with DNase I digestion in the extraction columns. RNA concentration was measured using Qubit 2.0 Fluorometric Quantitation (Life Technologies), and the RNA integrity number was determined using Experion Automated Electrophoresis System (Bio-Rad). RNA-seq libraries were prepared using a Sciclone NGS Workstation (PerkinElmer) and a Zepyhr NGS Workstation (PerkinElmer) with the TruSeq Stranded mRNA LT sample preparation kit (Illumina). Library amount and quality were determined using Qubit 2.0 Fluorometric Quantitation (Life Technologies) and Experion Automated Electrophoresis System (Bio-Rad). The libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq 3000/4000 platform and the 50-bp single-read configuration.

Sorted naive CD4⁺ T cells were cultured under Th0 conditions in the presence of rhIL-2 as described above. After 3 days, cells were harvested and stained with 7-AAD for 5 minutes, and alive cells were purified by FACS sorting. Total RNA was prepared from approximately 2 × 10⁶ cells and isolated using RNeasy Mini Kit (QIAGEN Inc.), including an on-column DNA digestion step (RNAse-Free DNAse Set, QIAGEN Inc.). RNA amount was measured using Qubit 2.0 Fluorometric Quantitation (Life Technologies), and RNA integrity number was determined using Experion Automated Electrophoresis System (Bio-Rad). RNA-Seq libraries were generated using a Sciclone NGS Workstation (PerkinElmer) and a Zepyhr NGS Workstation (PerkinElmer) with the TruSeq Stranded mRNA LT sample preparation kit (Illumina). Library amount and quality were determined using Qubit 2.0 and Automated Electrophoresis System (Bio-Rad). The libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq 3000 platform and the 50-bp single-read configuration. The RNA-Seq data have been deposited in the National Center for Biotechnology Information’s Gene Expression Omnibus database under number GSE134368.