title: GSM4478746: FA19 cells, Glucose 1 mM rep1; Neisseria gonorrhoeae; RNA-Seq
library_construction_protocol: Total RNA extraction was conducted using the RNeasy mini kit (Qiagen), following the manufacturer's protocol. One hundred µL of 200 ng/µL total isolated RNA were treated to remove genomic DNA contamination using the Turbo DNA-free Kit (Invitrogen, Thermo Fisher Scientific) as per the manufacturer's instructions. One hundred µL of DNase I-digested total RNA samples were concentrated and cleaned-up using the RNeasy MinElute Cleanup Kit (QIAGEN). Finally, samples were eluted from the RNeasy MinElute spin columns with 26 µL of Nuclease-Free water (Ambion, Thermo Fisher Scientific) and stored at -80 °C until library preparation. Library construction and sequencing was done at the UAB Heflin Center for Genomic Sciences, Birmingham, AL. RNA-sequencing was performed on the Illumina NextSeq 500 as described by the manufacturer (Illumina Inc., San Diego, CA). The quality of the total RNA was assessed using the Agilent 2100 Bioanalyzer. RNA with a RNA Integrity Number (RIN) of 7.0 or above was used for sequencing library preparation. Library preparation was done using the Agilent SureSelect Strand Specific mRNA library kit as per the manufacturer's instructions (Agilent, Santa Clara, CA). Library construction began with ribosome reduction using the RiboMinus rRNA depletion kit for bacteria (Invitrogen). The resulting RNA was randomly fragmented with cations and heat, which was followed by first strand synthesis using random primers with inclusion of Actinomycin D (2.4ng/µL final concentration). Second strand cDNA production was done with standard techniques, the ends of the resulting cDNA were made blunt, A-tailed and adaptors ligated for amplification and indexing to allow for multiplexing during sequencing. The cDNA libraries were quantitated using qPCR in a Roche LightCycler 480 with the Kapa Biosystems kit for Illumina library quantitation (Kapa Biosystems, Woburn, MA) prior to cluster generation.  Cluster generation was performed according to the manufacturer's recommendations for onboard clustering (Illumina).
attributes.tag: GEO Accession
attributes.value: GSM4478746