GSM9830537: R1203, before selection; Escherichia coli; RNA-Seq
The assembled products were mixed with electrocompetent cells and transformed into electrocompetent cells in a 2-mm Gene Pulser cuvette (Bio-Rad, 165-2086) using an electroporation instrument (Bio-Rad, MicroPulser). The transformants were cultured on 20 mL LB for 1 h at 37 °C on a rotator with 220 rpm. After 1 h, the size of the constructed library was detected by the number of colonies after a gradient dilution on LB-agar plate (containing 100 μg/mL ampicillin). Then 100 μg/mL ampicillin was added to 20 mL LB and allowed to grow at 37 °C for 10 h and collected using centrifugation. The pellet was resuspended in LB medium containing 15% glycerol and stored at -80 °C for long term use. 10^7 clones from the mutated library were cultured and amplified. The corresponding plasmids were extracted. The BspQ I linearized vectors were transcribed to RNAs. The purified RNA was reverse transcribed using ProtoScript® II Reverse Transcriptase (NEB, M0368L). The 20 μL reaction volume containing 4 µL 5 × ProtoScript II buffer, 2 μL 0.1 M DTT, 1 μL 10 mM dNTP, 0.2 μL RNase inhibitor (40 U/μL), 1 μL ProtoScript II RT (200 U/µL), 2 μL M13-RP and the RNA template. The reaction system was incubated at 42 °C for 1 h and then inactivated at 65 °C for 20 min. The cDNA product was stored at -20 °C for further use. A 10 μL cDNA product was utilized as the template for amplification (10 cycles) of the fragments with primers M13-FP/M13-RP. Then, the DNA sequence library was constructed using recycled fragments as the template with adaptor primers in PCR amplification reactions. The PCR products were purified by agarose gel electrophoresis with a Gel Extraction Kit (Omega, D2500-02).