NextSeq 500 sequencing; RNA-seq of Streptococcus thermophilus N4L grown in two different yeast extracts
RNA-seq of Streptococcus thermophilus N4L grown in two different yeast extracts
Samples were collected after 3, 4 and 5 hours of growth in yeast extract-based growth medium. After harvest, supernatants were removed by centrifugation (18,000 x g 15 sec) and bacterial pellets were frozen in liquid nitrogen. Streptococcus thermophilus N4L was grown in 1L-fermenters with the following parameters: : temperature of 40°C, agitation of 50 rpm, initial pH manually adjusted to 6.6 before inoculation, then automatically regulated at 6.0 with 2 M sodium hydroxide, and nitrogen supply at 0.2 l/min in the headspace. The culture medium contained (w/v) 6% lactose, 0.01% calcium chloride, 2% yeast extract, and a pool a vitamins as previously described (PMID: 11851809). Two distinct yeast extracts were used (YE1 and YE2). Cells were lysed in phenol-chloroform 5:1 (v/v) with a FastPrep® FT120 (Thermo Savant, USA). Two cycles of cell disruption were performed, both times at 6.5 m/sec for 45 sec. Cellular debris was pelleted by centrifugation (16,000 × g, 10 min, 4°C) and the RNA-containing aqueous supernatants were collected. Total RNA extraction was then performed using the Direct-Zol™ RNA MiniPrep kit (ZymoResearch, USA) according to the manufacturer instructions. Contaminant genomic DNA was removed with the DNA-free™ DNA Removal kit (Invitrogen, USA).  Purified RNAs were then submitted to an agarose gel-based migration in order to separate short transcripts (≈20-200 nt) from regular transcripts (> 200 nt). The latter were depleted of ribosomal RNA using the Ribo-Zero rRNA removal kit (Epicentre, USA). Finally, assessment of RNA quantity and quality of both preparations (short and regular transcripts) was achieved using a Qubit® Fluorometer (ThermoFisher, USA) and a Bioanalyzer system (Agilent, USA), respectively.
Experimental Factor: compound
yeast extract 2
Experimental Factor: time
4
Experimental Factor: fraction
long transcripts (regular)