title: GSM1405440: C1TLV30_26MPa_rep2; Pseudodesulfovibrio piezophilus C1TLV30; RNA-Seq
library_construction_protocol: For mRNA enrichment, 23S and 16S rRNA were depleted using a Ribo-ZeroTM Magnetic Kit (Gram-Negative Bacteria by Epicentre, Illumina®, Cat. No. MRZGN126), according to the manufacturer's instructions. Briefly, 5 µg of total RNA of each sample was treated by Ribo-Zero rRNA Removal Solution; rRNA was then removed using magnetic beads from Ribo-ZeroTM Magnetic Core Kit (Cat. No. MRZ116C). The ribo-depleted samples were purified by ethanol precipitation and eluted in 13 µl of RNase Free water. The concentration of resulted RNA was determined using NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), between 8-16 % of the input RNA was recovered after purification. RNA integrity was assessed on the Agilent 2100 Bioanalyzer with the RNA 6000 Pico chip Kit. Strand-specific libraries constructions were carried out using the SOLiD Total RNA-Seq Kit (Life Technologies) as describes in the manufacturer's instructions. About 0.35 µg of ribo-depleted RNA was subjected to the enzymatic mRNA fragmentation using RNaseIII from the SOLiD Total RNA-Seq Kit. The fragmented RNA was then concentrated using the Invitrogen RiboMinusTM Concentration Module according to the manufacturer's protocol. The quantity and quality of resulted RNA were assessed using a Qubit Fluorometer (Life Technologies) and Agilent 2100 Bioanalyzer with the RNA 6000 Pico chip Kit.  Fragmented RNA (0.1 µg) was subjected to cDNA synthesis. After hybridization, ligation of the SOLiDTM Adaptator, RNA was reversed transcripted at 42 °C for 30 min. The resulted cDNA was purified using the Agencourt AMPure XP Reagent performing two rounds. The eluted cDNA was then amplified by PCR reaction (10 cycles: 95 °C 5 min, 95 °C 30 sec, 62 °C 30 sec, 72 °C 30 sec, 72 °C 7 min) using a SOLiDTM 5' primer and a SOLiDTM 3' primer specific for each sample (barcode allowing us to pool the samples for sequencing), the resulted product was then purified using the same technique used for the cDNA purification (Agencourt AMPure XP Reagent). The quality and integrity of this DNA was confirmed using a NanoDrop ND-1000 Spectrophotometer and Agilent 2100 Bioanalyzer with DNA 1000 Kit. cDNA libraries were constructed from two independent experiments, for each pressure growth condition. The libraries were amplified with the SOLiD EZ Bead Amplifier, before that the samples were prepared using the SOLiD EZ Bead Emulsifier that allow mixing the aqueous phase containing the SOLiD EZ Bead, library template and primers with the oil phase (Oil Master Mix). The preparation was then amplified by PCR reaction (amplifier software scale E80). After the run was complete, the samples were purified using the SOLiD EZ Bead Enricher according to the manufacturer's instructions. The P2-enriched beads were then concentrated and the 3'ends were modified as described in the manufacturer's protocol. Paired-end stranded RNA-Seq with ribo-depleted total RNA was performed using a 5500xl SOLiD high-throughput sequencer (Life Technologies).
attributes.tag: GEO Accession
attributes.value: GSM1405440