RNA-seq of human: CD4+ T cell
RNA-seq libraries were prepared from 10 ng samples of RNA using the Illumina SmartSeq2 protocol. During reverse transcription, free dNTPs and oligo(dT) primers are used, which results in the addition of 2–5 non-templated nucleotides to the 3′ end of the cDNA. Next, a template-switching oligonucleotide (TSO) that carries two riboguanosines and a locked nucleic acid (LNA)-modified guanosine at the 3' end is introduced. The first-strand cDNA is then selectively amplified using a limited number of PCR cycles. After amplification, tagmentation is applied to fragment the cDNA and to add adapter sequences, which are necessary for next-generation sequencing.