GSM6637255: CBDS_Serum_3; Homo sapiens; miRNA-Seq
Add 700 μL QIAzol Lysis Reagent to the exosome's suspension. Mix by vortexing 10 s. Centrifuge for a short time and leave at room temperature for 5min.  Add 140 μL of chloroform/isoamyl alcohol (24:1), invert vigorously up and down for 15 s, and incubate for 2~3 min at room temperature.Centrifuge in 12,000×g for 8min in 4℃. Transfer the supernatant to a new tube. Add ethanol absolute two times of an equal volume to the supernatant, mix thoroughly. Pipet up to all sample into a spin column in a 2 mL collection tube and discard the flow-through.  Wash the column with 500 µL Buffer RPE.  Wash the column with 500 µL Buffer RPE.  Place the RNeasy MinElute spin column into a new 2 mL collection tube. Centrifuge at 12,000×g for 2 min to dry the membrane. Discard the collection tube with the flow-through. Place the RNeasy MinElute spin column in a new 1.5 mL collection tube. Add 20 µL RNase-free water directly to the center of the spin column membrane. Incubate for 1min at room temperature and centrifuge for 1 min at full speed to elute the RNA. Select the corresponding testing methods for quality inspection according to the requirements of samples and products. Take a certain amount of RNA sample, purified 18-30nt small RNA with PAGE gel. The purified RNA is combined with 3' Adapter, and incubated. Then combine 5' Adapter, and incubate. Prepare the Reverse Transcription Reaction Mixture, into the thermocycler and run the program. Prepare the PCR Reaction Mixture, into the thermocycler and run the program.Then the PCR products are purified with PAGE gel, dissolve the recylced products in EB solution.The corresponding library quality control protocol will be selected depending upon product requirements.Single-stranded PCR products are produced via denaturation. The reaction system and program for circularization are subsequently configured and set up. Single-stranded cyclized products are produced, while uncyclized linear DNA molecules are digested.Single-stranded circle DNA molecules are replicated via rolling cycle amplification, and a DNA nanoball (DNB) which contain multiple copies of DNA is generated. Sufficient quality DNBs are then loaded into patterned nanoarrays using high-intensity DNA nanochip technique and sequenced through combinatorial Probe-Anchor Synthesis (cPAS).