GSM9076966: CHI 12.5 mg/mL; Mus musculus; RNA-Seq
The tissue is washed with preconfigured PBS solution, sterilized mechanically spliced, and digested with preconfigured digestion solution for 30-60 minutes (depending on the type of tissue). After digestion is terminated, centrifugation, supernatant removal, and resuspension are performed. The suspension is collected by centrifugation through a 70 nm sieve and subjected to cell lysis red (depending on the type of tissue, lysis red treatment is selected). After lysis red is completed, centrifugation and resuspension are performed through a 40 nm sieve. Finally, counting cell number and viability to observe and judge whether the cell suspension was qualified. After collection of cells, centrifuge at a speed not exceeding 400rcf (adjust the speed and centrifugation time according to the cell types). Carefully discard the supernatant and retain the cell precipitation. The cell pellet was resuspended by 1mL 1x PBS containing 0.04% BSA and the washing procedure was repeated twice. After washing, appropriate volume PBS was added to the cell precipitation to obtain single-cell dispersion suspension with a concentration close to the goal number. A type of wide-bore pipette tip was used in cell resuspension for a lower cell damage. If necessary, cell filter should be applied to remove cell debris and clusters (eg.30µm MACS SmartStrainers and 40µm FlowmiTM Cell Strainer). Automatic cytometry was used to determine the cell concentration. And the sample volume was calculated based on the optimal cell sampling concentration supplied by 10X official website and the target capture number. If the calculated concentration is too high, adjust the liquid volume to the appropriate concentration and repeat counting once more. Once the desired cell suspension was obtained, immediately place the samples on ice for subsequent GEMs preparation and reverse transcription. After cell counts and viability were determined, cell suspensions were adjusted to a concentration of 1,000 cells/ml in PBS supplemented with 0.04% BSA. The 10X Genomics Chromium Next GEM Single Cell 3' Kit v3.1 was used according to the manufacturer's instructions. Agilent 4200 TapeStation was used to detect the fragment length distribution of the library. Also, Qubit HS DNA Kit was used to accurately quantify the effective concentration of the library ( > 10nmol/L).The qualified library was sequenced on the DNBSEQ T7 platform at the illumina NovaSeq 6000 with PE150 read length.