GSM4728010: CJ_CG84-21_dRNA_R2_plus_TEX; Campylobacter jejuni subsp. jejuni CG8421; RNA-Seq
Total RNA was purified using Hot-Phenol RNA isolation method from C.jejuni CG84-21 cells and used for TEX treatment. Briefly, total RNA isolated from CG84-21 was treated with DNase I, and equal amounts of Campylobacter RNA were incubated with Terminator™ 5′-phosphate-dependent exonuclease (TEX) (Epicentre, cat. #TER51020) for depletion of processed transcripts as previously described(2)(9). cDNA libraries for Illumina sequencing were then constructed by Vertis Biotechnologie AG, Germany (http://www.vertis-biotech.com) in a strand-specific manner (2)(9). In brief, RNA samples were poly(A)-tailed using poly(A) polymerase. Then, 5′-triphosphates were removed using tobacco acid pyrophosphatase (TAP), and an RNA adaptor was then ligated to the resulting 5′-monophosphate. First-strand cDNA was synthesized with an oligo(dT)-adaptor primer using M-MLV reverse transcriptase. A PCR-based amplification step was then conducted with a high-fidelity DNA polymerase to increase the cDNA concentration to 10-20 ng/μL. For all libraries, the Agencourt AMPure XP kit (Beckman Coulter Genomics) was used to purify the DNA, which was subsequently analyzed by capillary electrophoresis.  A library-specific barcode for multiplex sequencing was included as part of a 3′-sequencing adaptor. The following adaptor sequences flank the cDNA inserts:  TruSeq_Sense_primer:  5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′  TruSeq_Antisense_NNNNNN_primer:  5′-CAAGCAGAAGACGGCATACGAGAT-NNNNNN-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC(dT25)-3′  (NNNNNN = 6nt barcode for multiplexing):