GSM9011774: TMD8, C, rep1; Homo sapiens; RNA-Seq
1.1 Cell Sample Lysis Add appropriate TRIzol lysis buffer into the cell sample tube (1 mL TRIzol for 1×106 cells). 1.2 Sample Purification 1.2.1 Centrifuge the tissue samples after grinding and crushing at 12000×g for 5 min in 4℃. 1.2.2 Transfer the supernatant to the centrifuge tube with 300 µL Chloroform/isoamyl alcohol (24:1) mix thoroughly by upside down violent shaking； 1.2.3 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). 1.2.4 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. 1.2.5 Centrifuge at 17500×g for 25 min in 4℃. Discard the supernatant, wash with 0.9 mL 75% ethanol, suspend the precipitation by inverting. 1.2.6 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℃)。 1.2.7 Discard the supernatant, centrifuge for a short time and use the tip to remove the residual liquid, air dry for 3-5 min. 1.2.8 Dissolve the precipitation with 20-200 μL DEPC-H2O or RNase-free water. 2.mRNA Library Preparation 2.1 Sample QC Select the corresponding testing methods for quality inspection according to the requirements of samples and products. 2.2 mRNA Isolation A certain amount of RNA samples are denatured at suitable temperature to open their secondary structure, and mRNA is enriched by oligo (dT) -attached magnetic beads. 2.3 mRNA Fragmentation The reaction system is configured. After reacting at the suitable temperature for a fixed period of time, RNAs are fragmented. 2.4 cDNA Synthesis Prepare the first-strand synthesis reaction system, and set up the reaction program, synthesize the first- strand cDNA, prepare the second-strand synthesis reaction system, and set up the reaction program to synthesize the second-strand cDNA. 2.5 End Repair, Add A and Adaptor Ligation After the reaction system and program are configured and set up, double-stranded cDNA fragments are subjected to end-repair, and then a single 'A' nucleotide is added to the 3' ends of the blunt fragments. The reaction system and program for adaptor ligation are subsequently configured and set up to ligate adaptors with the cDNAs. 2.6 PCR The PCR reaction system and program are configured and set up to amplify the product. 2.7 Library QC The corresponding library quality control protocol will be selected depending upon product requirements. 2.8 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. 2.9 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).