GSM8118261: B12A, invasive; Homo sapiens; RNA-Seq
2.1 Tissue Sample Fragmentation and Lysis 2.1.1 Grinding Machine 2.1.1.1 Transfer appropriate sample to corresponding grinding tube, 2.1.1.2 Add 1.5 mL TRIzol lysis buffer, transfer to the grinding machine, grind 30 s, stay at 5 min 2.1.2 Liquid Nitrogen Grinding and Crushing 2.1.2.1 Dispense 1.5 mL of TRIzol lysis buffer to a 2 mL tube. 2.1.2.2 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. 2.1.3 Cell Sample Lysis Add appropriate TRIzol lysis buffer into the cell sample tube (1 mL TRIzol for 1×106 cells) 2.2 Sample Purification 2.2.1 Centrifuge the tissue samples after grinding and crushing at 12000×g for 5 min in 4℃. 2.2.2 Transfer the supernatant to the centrifuge tube with 300 µL Chloroform/isoamyl alcohol (24:1) mix thoroughly by upside down violent shaking； 2.2.3 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). 2.2.4 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. 2.2.5 Centrifuge at 17500×g for 25 min in 4℃. Discard the supernatant, wash with 0.9 mL 75% ethanol, suspend the precipitation by inverting. 2.2.6 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℃)。 2.2.7 Discard the supernatant, centrifuge for a short time and use the tip to remove the residual liquid, air dry for 3-5 min. 2.2.8 Dissolve the precipitation with 20-200 μL DEPC-H2O or RNase-free water. The raw data obtained from sequencing was filtered using SOAPnuke (v1.5.6), which removed the following: 1) reads containing adapters (adapter contamination); 2) reads with an unknown base N content greater than 5%; and 3) low-quality reads.