NextSeq 500 sequencing; Beta-glucan is a major growth substrate for human gut bacteria related to Coprococcus eutactus
Beta-glucan is a major growth substrate for human gut bacteria related to Coprococcus eutactus
Triplicate cultures of Coprococcus sp. ART55/1 grown were harvested during exponential phase (optical density 0.45-0.84). An aliquot of 0.5 ml culter was combined with 1 ml of RNA protect solution, vortexed for 5 seconds, incubated at room temperature for 5 minutes and centrifuged for 10 minutes at 5000g at room temperature. The supernatant was decanted and the cell pellet frozen at -70 degrees celsius until RNA extraction.  Overnight cultures grown in YCFA medium containing 0.2% of glucose as a carbohydrate source were inoculated into YCFA medium containing 0.2% of one of the following carbohydrates: cellobiose, barley beta-glucan, lichenan, glucomannan, or glucose.  The pellet was washed once with 50 mM sodium phosphate buffer, pH 6.5. The supernatant was removed, the cell pellet was resuspended in 0.5 ml reswell 3-10 buffer (7M urea, 2M thiourea, 4% CHAPS, 0.3% DTT) and 0.125 ml of 106 um glass beads suspended in water. Bead-beating was performed with a FastPrep machine, six times for 10 seconds on maximum setting with a minimum of 30 seconds on ice in between. The samples were centrifuged (max speed, at 4 degress celsius, 10 minutes). The RNA was then extracted using XXX kit as per the manufacturer's instructions.  Ribosomal RNA depletion was carried out utilising Ribo-Zero rRNA Removal Kit (Gram-Positive Bacteria) to the manufacturer's specification. Libraries were prepared with the Illumina TruSeq Stranded mRNA Library Preparation Kit.
Experimental Factor: growth condition
0.2 percent glucomannan