title: GSM7917905: NC3; Homo sapiens; RNA-Seq
library_construction_protocol: 1. Prepare enzyme digestion solution according to 2mg/ml collagenase I, 1mg/ml collagenase II, 0.04 mg/ml DNase I, 60 U/ml hyaluronidase and HBSSand filter it;2. Pour the sample tissue into the petri dish, place it on the ice plate, record the number, take pictures for retention, add the cleaning solution, and clean it twice with the cleaning solution;3. After discarding the cleaning solution, add a small amount of digestive solution to the petri dish and cut the sample tissue with scissors;4. After the tissue is cut, rinse the scissors with 1ml digestive liquid, collect and transfer to a suitable centrifuge tube, and add appropriate amount of enzyme liquid.5. 20μl digestive suspension was taken for Trypan blue staining microscopy.6. Seal the centrifuge tube with a sealing film and place the sample in a shaking table at 37ºC for 1h digestion;7. Blow the digestive fluid after digestion for 30 minutes, observe the digestive effect, and find that the tissue is basically digested8. After digestion, 20μl digestive suspension was taken for Trypan blue staining microscopy.9. Transfer the digestive fluid through the 100μm and 40μm filter to a 50ml centrifuge tube and centrifuge 300×g for 5min;10. Discard the upper liquid, add 1ml erythrocyte lysate to gently blow the cells, leave for 3min, transfer to 15ml tube;11. Add cleaning solution to 10ml to stop cracking red, centrifuge 300×g for 5min;12. The supernatant was discarded, the cleaning solution was added and re-mixed, and 20μl was taken respectively for AO/PI counting and Trisan blue microscopy. The results were reported. Sequencing libraries constructed with reagents from a Chromium Single Cell 3' v2(v2 or v3, depends on project) reagent kit (10X Genomics) according to the manufacturer's instructions.