title: GSM2992350: WT-1; Pseudomonas fluorescens; RNA-Seq
library_construction_protocol: The 10 ml culture was mixed with 20 ml RNAprotect Bacteria Reagent (Qiagen) and incubated for 20 min at room temperature to stabilize the mRNA. Next, the cell suspensions were centrifuged at 8,000 × g for 5 min to pellet the cells, and the supernatant was decanted. Total RNA was purified using RNeasy Mini Kit (Qiagen) according to the manufacturer's recommended protocol After the total RNA extraction and DNase I treatment, mRNA was isolated by removing rRNAs from the total RNA. Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments. Then cDNA is synthesized using the mRNA fragments as templates. Short fragments are purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. After  that, the short fragments are connected with adapters. After agarose gel electrophoresis, the suitable fragments are selected for the PCR amplification as templates. During the QC steps, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System are used in quantification and qualification of the sample library. At last, the library could be sequenced using Illumina HiSeqTM 2000 or other sequencer when necessary.
attributes.tag: GEO Accession
attributes.value: GSM2992350