GSM8893068: WT_SPI2_rep2; Salmonella enterica; OTHER
Trizol RIL-seq library preparation was conducted using the sRNA NEBNext kit for Illumina. 3 µL of RNA were mixed with 1 µL of 3' SR Adaptor (1:10 diluted) and incubated at 70°C for 2 min. 6.5 µL of 3' ligation mix (5 µL of 3' ligation buffer, 1.5 µL of 3' enzyme mix) were added and incubated at 25°C for 1 h. 2.75 µL of SR mix (2.5 µL of water, 0.25 µL of SR RT primer) were added to reactions and incubated for three subsequential steps (75°C for 5 min, 37°C for 15 min, 25°C for 15 min). Pre-denatured 5' adaptor (1:10 diluted) was added to each tube together with 5' ligation mix (0.5 µL of 5' ligation reaction buffer, 1.25 µL 5' ligation enzyme mix) and samples were incubated at 25°C for 1 hour. For first-strand cDNA synthesis, 5 µL of cDNA mix were added (4 µL first-strand buffer, 0.5 µL murine RNase inhibitor, 0.5 µL SuperScript II RT). The reactions were incubated at 50°C for 1 h followed by 15 min at 70°C. 10 µL of cDNA were PCR-amplified with barcoded NEB index primer and SR primer in a 50 µL reaction (25 µL LongAmp Taq 2x mix, 12.5 µL nuclease-free water, 1.25 µL SR primer, 1.25 µL index primer).