GSM8374645: Ovary miRNA, Year 1, rep3; Sus scrofa; miRNA-Seq
Small RNA Library Construction Protocol 1 Sample QC Select the corresponding testing methods for quality inspection according to the requirements of samples and products. 2 Adapter Ligation Take a certain amount of RNA sample,The purified RNA is combined with 3' Adapter, and incubated. Then combine 5' Adapter, and incubate.  3 RT - PCR 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. 4 Purify PCR products Then the PCR products are purified with PAGE gel, dissolve the recylced products in EB solution. 5 Library QC The corresponding library quality control protocol will be selected depending upon product requirements. 6 Circularization 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. 7 Sequencing 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). 1 Tissue Sample Fragmentation and Lysis 1.1 Grinding Machine 1.1.1 Transfer appropriate sample to corresponding grinding tube, 1.1.2 Add 1.5 mL TRIzol lysis buffer, transfer to the grinding machine, grind 30 s, stay at 5 min 1.2 Liquid Nitrogen Grinding and Crushing 1.2.1 Dispense 1.5 mL of TRIzol lysis buffer to a 2 mL tube. 1.2.2 Take appropriate amount of tissue samples, grind the tissue samples into powder with liquid nitrogen and transfer into the lysis buffer. Stand flat for 5 min for the fully lysis of tissue cells. 1.3 Cell Sample Lysis Add appropriate TRIzol lysis buffer into the cell sample tube (1 mL TRIzol for 1×106 cells) 2 Sample Purification 2.1 Centrifuge the tissue samples after grinding and crushing at 12000×g for 5 min in 4℃. 2.2 Transfer the supernatant to the centrifuge tube with 300 µL Chloroform/isoamyl alcohol (24:1) mix thoroughly by upside down violent shaking； 2.3 Centrifuge at 12000×g for 8 min in 4℃ (optional extraction with equal supernatant volume of chloroform/isoamyl alcohol (24:1) could be used if the middle layer is thick and the water phase turbidity). 2.4 Transfer the supernatant to the 1.5 mL centrifuge tube, add 2/3 volume of Isopropyl alcohol, (optional 2µL 5 mg/mL glycogen for low-input tissue and cell sample to help precipitation). Gently invert and mix, place in -20℃ refrigerator for more than 2 h. 2.5 Centrifuge at 17500×g for 25 min in 4℃. Discard the supernatant, wash with 0.9 mL 75% ethanol, suspend the precipitation by inverting. 2.6 Centrifuge at 17500×g for 3 min in 4℃ (optional wash with 0.9 mL 75％ ethanol could be used, 17500×g for 3 min in 4℃)。 2.7 Discard the supernatant, centrifuge for a short time and use the tip to remove the residual liquid, air dry for 3-5 min. 2.8 Dissolve the precipitation with 20-200 μL DEPC-H2O or RNase-free water.