RNA-Seq of K. radicincitans: amtB gene deletion mutant of GXGL-4A
The total RNA was extracted using TRIzol reagent according the manufacturers instructions (Invitrogen Corp., Carlsbad, CA). Genomic DNA was removed using DNase I (TaKara, Japan). RNA quality was analyzed by an Agilent 2100 Bioanalyzer (Agilent Technologies, Pale Alto, Calif.) and the RNA concentration was measured on a NanoDrop ND-2000 (Thermo Fisher Scientific, Waltham, Mass.). RNA-seq library was constructed according to the manual of the TruSeqTM RNA sample preparation Kit from Illumina (San Diego, CA) by using 2 g of total RNA. The ribosomal RNA (rRNA) depletion instead of poly(A) purification was carried out using Ribo-ZeroTM Magnetic Kit (Epicenter, Madison, WI), and then all mRNAs were broken into short fragments (around 200 nt) by adding fragmentation buffer. Double-stranded cDNA was synthesized using a SuperScriptTM Double-Stranded cDNA Synthesis Kit (Invitrogen, CA) with random hexamer primers (Illumina). The synthesized cDNA was subjected to end-repair, phosphorylation and A base addition according to the Illuminas library construction protocol. Libraries were selected for cDNA target fragments of 200bp on a 2% Low Range Ultra Agarose followed by a PCR amplification (15 cycles) using Phusion DNA polymerase (NEB). The paired-end RNA-seq library was sequenced by 2150 bp double terminal sequencing strategy using an Illumina HiSeq X Ten sequencer after a quantification by TBS380.