Concentration Recognition-Based Auto-Dynamic Regulation System (CRUISE) Enabling Efficient Production of Higher Alcohols
Total RNA of each sample was extracted using TRIzol Reagent /RNeasy Mini Kit (Qiagen). Total RNA of  each sample was quantified and qualified by Agilent 2100/2200 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), NanoDrop (Thermo Fisher Scientific Inc.) and 1% agrose gel.  1g total RNA was used  for following library preparation.  Next generation sequencing library preparations were constructed  according to the manufacturers protocol.  The rRNA was depleted from total RNA using rRNA removal Kit.  The ribosomal depleted RNA was then fragmented and reverse-transcribed.  First strand cDNA was  synthesized using ProtoScript II Reverse Transcriptase with random primers and Actinomycin D. The  second-strand cDNA was synthesized using Second Strand Synthesis Enzyme Mix  include dACGTP/dUTP .  The purified double-stranded cDNA by beads was then treated with End Prep Enzyme Mix  to repair both ends and add a dA-tailing in one reaction, followed by a T-A ligation to add adaptors to  both ends.  Size selection of Adaptor-ligated DNA was then performed using beads, and fragments of ~400 bp (with the approximate insert size of 300 bp) were recovered.  The dUTP-marked second strand  was digested with Uracil-Specific Excision Reagent enzyme.  Each sample was then amplified by PCR  using P5 and P7 primers, with both primers carrying sequences which can anneal with flow cell to  perform bridge PCR and P5/P7 primer carrying index allowing for multiplexing.  The PCR products were  cleaned up using beads, validated using an Qsep100 (Bioptic, Taiwan, China), and quantified by Qubit3.0 Fluorometer (Invitrogen, Carlsbad, CA,USA).  Then libraries with different indices were multiplexed and loaded on an Illumina HiSeq/Novaseq  instrument according to manufacturers instructions (Illumina, San Diego, CA, USA) or a MGI2000  instrument according to manufacturers instructions (MGI, Shenzhen, China) .  Sequencing was carried  out using a 2x150 paired-end (PE) configuration;  image analysis and base calling were conducted by the HiSeq Control Software (HCS) + OLB + GAPipeline-1.6 (Illumina) on the HiSeq instrument image  analysis and base calling were conducted by the NovaSeq Control Software (NCS) + OLB + GAPipeline- 1.6 (Illumina) on the NovaSeq instrument image analysis and base calling were conducted by the Zebeacall on the MGI2000 instrument.