title: RNA-Seq of homo sapiens: adult male patient with FECD
design: The 5' RACE protocol represents a refined method for amplifying the 5'-ends of T-cell receptor mRNAs. It incorporates reverse transcription with template switching and two-step nested PCR with PCR suppression. A total of 25-100 ng of RNA was reverse transcribed using the SMARTer RACE 5'/3' Kit (Takara, Japan), with the use of a template switch oligonucleotide (TSO) containing a Unique Molecular Identifier (UMI) to facilitate the removal of PCR duplicates. The TSO was designed with sequences for primer annealing, UMI, a service sequence, and a riboG-tail, which facilitate effective template switching. Polymerase chain reaction (PCR) amplification was conducted in two stages. The initial stage employed an outer gene-specific primer (GSP) and a long step-out primer, while the subsequent stage utilised an inner GSP and a short library primer, with the objective of suppressing non-specific products. Two distinct combinations of gene-specific primers (GSPs) were employed to target specific transcript variants of SLC4A11. Equal amounts (in ng) of the amplicons from the same cDNA sample with GSP1 and GSP2 were combined and used for Illumina MiSeq library preparation. The resulting amplicons were purified and quantified prior to pooling for Illumina MiSeq library preparation. This protocol enhances the specificity and efficiency of transcript amplification, thereby facilitating comprehensive analysis of gene expression.