Protocols: C. difficile cells were cultivated in BHIS medium to mid log phase (OD = 0.3 - 0.6), 10 ml cells were collected by centrifugation at 5000 g. Then mailed to Shanghai Personal Biotechnology Cp, Ltd with dry ice. Total RNA was isolated using the Trizol Reagent (Invitrogen Life Technologies). Quality and integrity were determined using a NanoDrop spectrophotometer (Thermo Scientific) and a Bioanalyzer 2100 system (Agilent). Sequencing libraries were generated according to the following steps. Firstly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in an Illumina proprietary fragmentation buffer. First strand cDNA was synthesized using random oligonucleotides and Super Script II reverse transciptase. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and the enzymes were removed. After adenylation of the 3′ ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated to prepare for hybridization. To select cDNA fragments of the preferred 400-500 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter,Beverly, CA, USA). DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15 cycle PCR reaction. Products were purified (AMPure XP system) and quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent).
ENA first public
2022-01-01
ENA last update
2022-01-01
External Id
SAMEA11298872
INSDC center alias
Guizhou Medical University
INSDC center name
Guizhou Medical University
INSDC first public
2022-01-01T00:13:21Z
INSDC last update
2022-01-01T00:13:21Z
INSDC status
public
Submitter Id
E-MTAB-11180:CD630-2
broker name
ArrayExpress
genotype
wild type genotype
organism part
whole organism
sample name
E-MTAB-11180:CD630-2