title: CAGE
design: Step 1. Prepare Sample DNA for Cluster Generation  Combine the template DNA and 0.1 N NaOH. Vortex briefly to mix the template solution. Centrifuge the template solution to 280 xg for one minute. Incubate for 5 minutes at room temperature to denature the template into single strands. Transfer 20  l of denatured template to a tube containing 980  l of pre-chilled HT1 (Hybridization Buffer). Dilute the denatured DNA to titration series with pre-chilled hybridization buffer and dispense eight-strip tube.   Step 2. Cluster Generation  Set the denatured template DNA in eight-strip tube, and add reagents required for cluster generation and flow cell to The cBot fluidics device according to manufacturer's protocol with TruSeq SR Cluster Kit v3 cBot HS (Catalog # GD-401-3001). Operate the cBot fluidics device using the Illumina cBot software to perform the following reactions:  a. Hybridize template DNA-Hybridize template molecules onto the oligonucleotide-coated surface of the flow cell.  b. Amplify template DNA-Isothermally amplify the molecules to generate clonal DNA clusters.  c . Linearize-Chemically linearize the dsDNA clusters. This is the first step of converting dsDNA to ssDNA that is suitable for sequencing.  d. Block-Block the free 3'-OH ends of the linearized dsDNA clustersto prevent nonspecific sites from being sequenced. After this step, the flow cell is stable and can be stored.  e. Denature-Convert the dsDNA to ssDNA.  f. Hybridize sequencing primers-Hybridize a sequencing primer, or multiple sequencing primers, onto the linearized and blocked clusters. After this step, the flow cell is ready for sequencing.   Step 3. Sequencing  After the completion of the above step, the lane [lane] of the flow cell (using flow cell v3) is used for sequencing with four-color DNA Sequencing-By-Synthesis (SBS) technology using the illumina(R) HiSeq 2000, [machine]. The sequencing run [run] and the base call analysis are performed according to the manufacturer's protocol with TruSeq SBS kit v3-HS (Catalog # FC-401-3001).   Step 4. Basecall  After the sequencing, sequence raw data is generated with processing by [software] with version [base_caller_version].