Thermus aquaticus Term-Seq Replicate 2
From the total RNA samples, ribosomal RNA molecules were depleted using an in-house developedprotocol. First, an adenylated oligonucleotode (5' Illumina sequencing adapter) was ligated to the 3'OH ends ofthe rRNA depleted RNAs. Then, first-strand cDNA synthesis was performed using M-MLV reversetranscriptase. The first strand cDNA was fragmented and to the 3'-ends of the ss-cDNA fragments the3' Illumina sequencing adapter was ligated. Finally, the 3' cDNA fragments were PCR-amplified usinga high fidelity DNA polymerase. The cDNA was purified using the Agencourt AMPure XP kit (BeckmanCoulter Genomics) and analyzed by capillary electrophoresis. The cDNA pools were sequenced on an Illumina NextSeq 500 system using 1x75 bp read length. Inthe case of 3Term samples, reads are in antisense orientation and pass through the 3'-ends of theRNA fragments!