GSM2817103: SF3+TEX; Vibrio harveyi NBRC 15634 = ATCC 14126; RNA-Seq
Cell pellets were collected by centrifugation, total RNA was extracted with TRIzol reagent (Ambion) and PureLink RNA Mini Kit (Ambion) and treated with DNaseI (Invitrogen) Fragmented RNA was poly(A)-tailed using poly(A) polymerase and an RNA adapter was ligated to the 5'-phosphate of the RNA. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and M-MLV reverse transcriptase. The resulting cDNA was PCR-amplified to about 10-20 ng/µl using a high-fidelity DNA polymerase. PCR cycles performed and barcode sequences, which are attached to the 5' ends of the cDNAs, are as follows: SF1-star AGGATAGG 13 cycles, SF2-star TCAGAGCC 13 cycles, SF3-star CTTCGCCT 13 cycles, SF1-star+TEX CTCCTTAC 15 cycles, SF2-star+TEX TATGCAGT 15 cycles, SF2-star+TEX TACTCCTT 15 cycles. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and analyzed by capillary electrophoresis. For Illumina NextSeq sequencing, the cDNA preparations were pooled in approximately equimolar amounts and size fractionated in the size range of 200 – 500 bp using a differential clean-up with the Agencourt AMPure kit. The primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina. The following adapter sequences flank the DNA insert: TruSeq_Sense_primer (N=barcode) AATGATACGGCGACCACCGAGATCTACAC-NNNNNNNN-ACACTCTTTCCCTACACGACGCTCTTCCGATCT, TruSeq_Antisense_primer CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC(dT)25
GEO Accession
GSM2817103