title: GSM2310616: HP26695 CrppH -TEX +TAP; Helicobacter pylori 26695; RNA-Seq
library_construction_protocol: Frozen cell pellets were thawed on ice and re-suspended in lysis solution containing 600 µl of 0.5 mg/ml lysozyme in TE buffer (pH 8.0) and 60 µl 10% SDS. Bacterial cells were lysed by incubating the samples for 1–2 minutes at 65°C. Afterwards, total RNA was extracted using the hot-phenol method described previously (Sharma et al. 2010, Bloomberg 1990, EMBO J). DNaseI (Fermentas) treatment was performed on total RNA according to manufacturer’s instruction. cDNA library preparation was performed by Vertis Biotechnology AG, Germany (http://www.vertis-biotech.com/) in a strand-specific manner. In brief, the three RNA samples were split into three portions. One portion was treated with Terminator exonuclease (TEX) prior to the standard library preparation procedure described below to generate the +TEX/+TAP libraries. To this end, RNA was denatured for 2 min at 90˚C, cooled on ice for 5 min and treated with 1.5 U of Terminator Exonuclease (Epicentre) for 30 min at 30˚C. For the second portion the TAP treatment (see below) was omitted to generate the −TEX/−TAP libraries. The standard procedure without modifications was used to generate the –TEX/+TAP libraries from the third portion. Here, ~200 ng of RNA sample were poly(A)-tailed using 2.5 U E. coli poly(A) polymerase (NEB) for 5 min at 37°C. Then, the 5'PPP structures were removed using tobacco acid pyrophosphatase (TAP). TAP treatment was performed by incubating the samples with 5 U TAP for 15 min at 37˚C. Afterwards, an RNA adapter (5' Illumina sequencing adapter, 5' UUUCCCUACACGACGCUCUUCCGAUCU 3') was ligated to the 5’P of the TAP-treated, poly(A)-tailed RNA for 30 min at 25°C. First-strand cDNA was synthesized by using an oligo(dT)-adapter primer and the M-MLV reverse transcriptase (AffinityScript, Agilent) by incubation at 42°C for 20 min, ramp to 55°C followed by 55°C for 5 min. In a PCR-based amplification step using a high fidelity DNA polymerase (Herculase II Fusion DNA Polymerases, Agilent) the cDNA concentration was increased to 20-30 ng/μl (initial denaturation at 95°C for 2 min, 14-16 cycles 95°C for 20 sec and 68°C for 2 min). A library-specific barcode for multiplex sequencing was included as part of a 3' sequencing adapter. The TruSeq index primers for PCR amplification were used according to the instructions of Illumina. For all libraries the Agencourt AMPure XP kit (Beckman Coulter Genomics) was used to purify the DNA (1.8 x sample volume), and cDNA sizes were examined by capillary electrophoresis on a MultiNA microchip electrophoresis system (Shimadzu).
attributes.tag: GEO Accession
attributes.value: GSM2310616