Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Frozen cell pellets were thawed on ice and resuspended in 600 µl lysis solution containing 0.5 mg/ml lysozyme in TE buffer, pH 8.0, and 60 µl 10% SDS. The cells were lysed by incubating the samples for 1-2 min at 64 °C. After the incubation, 1 M NaOAc, pH 5.2 (66 µl) was added and the sample was mixed by inversion. Total RNA was extracted using the hot-phenol method by adding 750 µl phenol. The solution was mixed by inversion and incubated for 6 min at 64 °C. Afterwards, the samples were mixed 6-10 times by inversion and cooled on ice. After centrifugation for 15 min at 14000 x g at 4 °C the aqueous layer was transferred and the chloroform extraction was performed in a 2 ml Phase Lock Gel tube (Eppendorf, Hamburg, Germany). 750 µl chloroform was added and mixed by inversion. After centrifugation for 12 min at 14000 x g at 15 °C, the aqueous layer was used for the ethanol precipitation. To the RNA containing sample, 0.1 volume 3 M NaOAc, pH 5.2 and two volumes of 96% ethanol (-20 °C) were added. The sample incubated for 2 h at -20 °C. Ethanol was discarded after centrifugation for 20 min at 14000 x g and 4 °C. The RNA was washed once with 200 µl -20 °C 70% ethanol. Ethanol was removed and the RNA was subjected to library preparation. The libraries were generated by Vertis Biotechnologie AG (Munich, Germany). In brief, the RNA samples were poly(A)-tailed using poly(A) polymerase. Terminator exonuclease treatment (+TEX) and mock treatment without the enzyme (-TEX) were carried out after poly(A)-tailing. In this way, corresponding cDNA pairs were generated. Then, the 5'PPP structures were removed using tobacco acid pyrophosphatase (TAP). Afterwards, an RNA adapter was ligated to the 5'-monophosphate of the RNA. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and the M-MLV reverse transcriptase. The resulting cDNAs were PCR-amplified to about 10-20 ng/μl using a high fidelity DNA polymerase. The cDNAs were purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and were analyzed by capillary electrophoresis. A library-specific barcode for multiplex sequencing was included as part of a 3'-sequencing adapter.