GSM4175949: WT rep3; Nostoc punctiforme PCC 73102; RNA-Seq
Triplicate cultures of log phase wild-type plasmid-only control (WTC) and 2g strains were harvested by a 2-minute centrifugation at 6,000xg. The pellet was suspended in 700 µl of media prior to flash freezing and storage at -80°C. RNA was harvested as previously described {Schmidt-Goff, 1993 #1370}. The samples were then further purified using a RNeasy Mini Kit (Qiagen) with on-column DNase treatment. Samples were aliquoted and stored at -80°C until used. 5 µg of each sample were cleaned and concentrated further using an RNA Clean & Concentrator -5 kit (Zymo). rRNA depletion was then performed using Terminator 5'-Phosphate-Dependent Exonuclease (Epicentre) following the manufacturer's protocol using Buffer A RNA libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina per the manufacturer's instructions. To determine the quality of the libraries, an Experion 1K DNA analysis kit (Bio-Rad) was used to assess the size distribution of the cDNA library following manufacturer's instructions. Each cDNA library was quantified using a Qubit Fluorometer and Qubit dsDNA High Sensitivity reagents (Invitrogen) using manufacturer's instructions, and also using qPCR following the protocol from the Library Quantification Kit for Illumina platforms (KAPA cat # 0796014000). Samples were normalized to 10 nM and multiplexed based on qPCR derived concentrations. The multiplexed library containing all 6 samples were sequenced on an Illumina HiSeq 2500 system where 100 bp single-end reads were generated (UC Irvine Genomics High Throughput Facility).
GEO
GSM4175949
GEO Accession
GSM4175949