GSM1439175
GSM1439175: TIE1_NF_rep_1; Rhodopseudomonas palustris; RNA-Seq
RNA was extracted and purified from 5 ml of the same cell suspensions and time points used for tracking the biomass composition. The R. palustris-specific selective primers v2 were based on the genome sequences of six R. palustris strains (CGA009, TIE-1, HaA2, BisB5, BisB18, and BisA53). We used a pool of 1,203 selective primers with no perfect match to any rRNA genes (e.g., 5S, 16S, and 23S rRNA). An additional set of 200 primers that were responsible for the majority of rRNA-priming events in test libraries, was further removed, leaving a final set of 1,003 R. palustris-specific selective primers v2 (Table S1). For first strand cDNA synthesis, 500 ng of total RNA was mixed with 1 μl of 100 μM of R. palustris-specific selective primers v2 in 5 μl reaction volume, and incubated at 65 °C for 5 min. After snap chilling on ice, 2 μl of 5X First-Strand Buffer (Invitrogen, Carlsbad, CA), 2 μl of 10 mM dNTP mix (New England BioLabs, Ipswich, MA), 0.5 μl of 0.1 M DTT, 0.5 μl of SuperScript III Reverse Transcriptase (Invitrogen) were added, and the mixture was further incubated at 40°C for 90 min. To generate the second strand, 45.5 μl of water, 15 μl of 5X Second Strand Buffer (Invitrogen), 1.5 μl of 10 mM dNTP mix, 0.5 μl of Escherichia coli DNA Ligase (Invitrogen, 10 U/μl), 2 μl of DNA Polymerase I (Invitrogen, 10 U/μl), and 0.5 μl of RNase H (Invitrogen, 2 U/μl) were added to the reaction, and incubated at 16 °C for 2 h. The reaction was stopped by adding 25 μl of 20 mM EDTA, pH8.0.  Double stranded cDNA was then purified by using the MinElute PCR Purification Kit (QIAGEN, Valencia, CA) using 12 μl of Buffer EB. Finally, the cDNA library was constructed using the Ovation Ultralow DR Multiplex System (NuGEN, San Carlos, CA) following the manufacture’s instructions.
GEO
GSM1439175
GEO Accession
GSM1439175