title: GSM8630586: ppGpp0 2; Pseudomonas putida; RNA-Seq
library_construction_protocol: Aliquots of 500 µL were harvested by centrifugation, frozen with liquid nitrogen and stored at -80 ºC. Cell pellets were resuspended in 1 mL of TRIsure™ (Bioline) and incubated for 10 min at 60 ºC. Samples were centrifuged and supernatants were transferred to Phase Lock Gel™ tubes (5PRIME). Each tube was added 200 µL of chloroform, mixed by inversion, kept in ice for 15 min and then centrifuged. Next, tubes were added 400 µL of phenol:chloroform:isoamyl alcohol (25:24:1) and then centrifuged. Aqueous phase was transferred to a new tube and 1 volume of isopropanol was added, mixed by inversion and kept in ice for 10 min. Tubes were centrifuged and pellets were washed with 1 mL of cold ethanol 70 % (v/v). Pellets were dried at room temperature and resuspended in RNAse-free water. Remaining DNA was degraded by treatment with TURBO™ DNase kit (Thermo Fischer) and purified using the RNAeasy Mini Kit (QIAGEN) following the manufacturers' instructions. Complete absence of DNA traces was checked by PCR. Samples containing 5 µg of total RNA were sent to the CABIMER Genomics Facility (Seville, Spain). Depletion of rRNA and preparation of cDNA libraries were carried out using the Illumina Stranded TOTAL RNA preparation RIBO-ZERO PLUS kit. Libraries were normalized to 0.5 nM, equimolar-mixed and sequenced using an Illumina® NextSeq™ 500 system with a Mid-Output flow cell to obtain 2 x 75 bp length paired-end reads. Libraries are fr-firstranded.