Whole Transcriptome Sequencing (RNA-Seq) of Rhizospheric Bacteria isolated from wheat plant
Whole Transcriptome Sequencing (RNA-Seq) of Rhizospheric Bacteria isolated from wheat plant
Samples were collected from rhizosphere of wheat plant Seher-2006. The plant was in booting stage when the rhizospheric bacteria were isolated. Rhizospheric bacteria were isolated from 1 g soil tightly adhering to the root by serial dilution plating on Luria–Bertani (LB) agar plates. Endophytic bacteria was isolated by serial dilution plating of sterilized crushed root samples on LB agar plates. The plates were incubated at 28 ± 2°C till the appearance of bacterial colonies. Individual colonies were picked and streaked on LB plates for further purification. RNA was extracted using kit method - GeneJET Plant RNA Purification Kit, Catalog number: K0801 1) Isolate the Total RNA from Sample of interest (Cell or Tissue). 2) Eliminate DNA contamination using DNase. 3) For mRNA with poly-A tail, mRNA purification kit was used; for non-coding RNAs, such as lincRNA was used. 4) Randomly fragment purified RNA for short read sequencing. 5) Reverse transcribe fragmented RNA into cDNA. 6) Ligate adapters onto both ends of the cDNA fragments. 7) After amplifying fragments using PCR, select fragments with insert sizes between 200-400 bp. For paired-end sequencing, both ends of the cDNA is sequenced by the read length.