title: GSM8679818: cftr+- larvae; Danio rerio; RNA-Seq
library_construction_protocol: The tissue samples was obtained from healthly donors and patients or mouse, the obtained tissue samples were washed 2-3 times in 4-degree precooled 1×PBS on ice, transferred to a sterile RNase-free culture dish , and cut into small pieces ( about 0.5mm2) with 10cm surgical scissors，the tissues were washed with 1×PBS, and remove as many non-purpose tissues as possible such as blood stains, fatty layers and connective tissue. The tissue samples were incubated in a constant temperature water bath or shaker at 37℃ with the self-prepared hydrolysate or the hydrolysate kit (MACS Human Tumor Dissociation kit DS_130-095-929/ MACS Mouse Tumor Dissociation kit DS_ 130-096-73 0). Generally, the incubation was terminated when the digestive juice was turbid and the tissue mass disappeared. Then Use 40µm cell sieve to filter, or use 100µm/70µm cell filter if the tissue mass is large. Generally, the cell suspension was centrifuged at 300r at 4℃ for 5min (Adjust centrifugal force and time according to cell size and number). Discard the supernatant and add 1mL of re-suspension to the centrifuge tube to re-suspend the cells, add 3mL of pre-cooled erythrocytes lysis liquid in tube (Solarbio), blowing and suction evenly. Incubate at 4℃ for 5-10min, centrifuge at 300r at 4℃ for 5min immediately after completion and remove the supernatant and add 1mL of re-suspension( Precooled 1X PBS) to fully re-suspend the cells. Test with cell counter（Countstar Rigel S2）, adjust the concentration according to the test result, the target concentration is 700-1200 cell/µL, the Cell activity is greater than 85%, and the agglomeration is less than 15%. Once the final cell concentration and activity were reached, the cells were placed on ice and 10x Genomics single cell transcriptome chip on-board experiment was carried out within 30 minutes. We prepared single cell RNA-seq libraries with Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 on the Chromium Controller(10× Genomics).We prepared single cell suspensions from cultured cell lines and single cells were suspended in PBS containing 0.04% BSA.The cell suspension was loaded onto the Chromium Next GEM Chip G and ran the Chromium Controller to generate single-cell gel beads in the emulsion(GEMs) according to the manufacturer's recommendation.captured cells were lysed and the released RNA was barcoded through reverse transcription in individual GEMs. Barcoded, full-length cDNA was generated and libraries were constructed according to the performer's protocol .The quality of libraries was assessed by Qubit 4.0 and the Agilent 2100. Sequencing was performed on the Illumina NovaSeq 6000 with a sequencing depth of at least 50,000 reads per cell and 150 bp (PE150) paired-end reads (performed by Biomarker Technologies Corporation, Beijing, China).