GSM2418922: CGATGT-D2; Escherichia coli; RNA-Seq
Samples were diluted 1:1 in RNA Protect (Qiagen, Carlsbad, CA) to inhibit RNase activity and then centrifuged (5000 x g, 10 min). Pellets were resuspended in TE/lysozyme (10 mg/ml lysozyme, 0.5% SDS, pH 8.0) with added proteinase K (1.5 mg/ml). RNA was isolated using Qiagen’s RNeasy kit according to manufacturer instructions with slight modification. Before centrifugation, β-mercaptoethanol was added to RNeasy kit buffer RLT (10% v/v). Additionally, during the wash step RNase-free DNase (Qiagen, Carlsbad, CA) was diluted in RNeasy kit buffer RDD (310 Kunitz units/mL), added to the purification column, and incubated for 15 minutes at room temperature (RT). After isolation, spectrophotometric NanoDrop (NanoDrop 1000 v3.8.1; Thermo Fisher) curves were obtained for each total RNA sample and verified for purity, as defined by absorbance ratios at 260/280 nm and 260/230 nm. Total RNA samples were sent to Oregon State University’s Center for Genome Research and Biocomputing. RNA integrity (RIN) measurements were taken using an Agilent Bioanalyzer, resulting in RIN scores of 10, out of a possible 10, for each sample. Ribosomal RNA was depleted using the RiboZero rRNA removal kit (Life Technologies, Eugene, OR). Resulting mRNA was reverse-transcribed to cDNA libraries via SuperScript III First Strand reverse transcription kit (Invitrogen, Carlsbad, CA, 18080051) as per the manufacturer’s instructions. The cDNA libraries were multiplexed to distinguish replicates from one another, barcoded for sequencing, and then amplified with random hexamers for 15 PCR cycles. Transcripts were sequenced for 50 bases in single-end fashion within one lane of an Illumina Hiseq 2000 flow cell. This yielded roughly 30 million reads per sample.
GEO Accession
GSM2418922