Gst40-Ps1_2
In the first step, adenylation of the 3-protected RNA adapter was performed using TS2126 ligase (CircLigase). Subsequently, this adapter was ligated to the 3-end of the native tRNAs using T4Rnl2KQ. The adapter introduced the index primer binding site and served as a binding site for the reverse transcription primer. The reverse transcription primer is modified with two hexa-ethylene glycol groups that prevent rolling circle amplification during final PCR. The reverse transcription primer is used to introduce the binding site for the sequencing primer. This was followed by reverse transcription, degradation of the RNA by increasing the pH, and size selection of the cDNA using PAA gels. The cDNA was then circularized and served as a template in the final PCR. The primers used here introduced both the Illumina Flowcell sequences P5 and P7 and one of 25 6 bp i7 indices into the library. After PCR, the libraries were purified using the QIAquick PCR Purification Kit.