RNAseq of homo sapiens: pediatric CD4+ T-cells: Sample A40_e
2.5 μg of RNA extracted from 2x106 unstimulated Th cells underwent directional RNA-seq based library preparation. Samples with RNA integrity number (RIN) of 8 or greater underwent processing, beginning with removal of ribosomal RNA with the Ribo-Zero rRNA removal kit (Illumina Inc., San Diego, CA). Next, reverse transcription was performed using the SuperScriptIII First-Strand Synthesis system, followed by second strand cDNA synthesis using dUTP (Thermo Fisher Scientific, Waltham, MA). The double stranded cDNA was fragmented with Covaris (200-300 bp target length), end-repaired, dA tailed, and adaptors added to allow for multiplexing of 8 samples per lane on the Illumina HiSeq 2500. To maintain directional information, i.e. transcribing strand-specific information, a combination of dUTP incorporation and uracil-DNA glycosylase were used. All libraries underwent Bioanalyzer testing for quality control and were sequenced on Illumina HiSeq 2500 as 100 bp single-end reads.
NHLBI-phs001812
A40_RNAseq