GSM2302445: A25; Homo sapiens; RNA-Seq
Ficoll hypaque method for PBMC separation followed by negative T cell isolation using magnetic beads Briefly, after removal of ribosomal RNA with the Ribo Zero-rRNA removal kit (Illumina Inc., San Diego, CA), reverse transcription was done using SuperSriptIII First-Strand Synthesis system followed by second strand cDNA synthesis using dUTP. The double stranded cDNA was fragmented with Covaris (200-300bp target length), end-repaired, dA tailed and adaptors added for the Illumina sequencer to allow multiplexing of 8 samples per lane. To maintain directional information, i.e. transcribing strand-specific information (PMID:24733242), 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 100bp single-end reads.