| 提取方案: |
Organoids were grown on a 6 well tissue culture dish for 18-30 days, washed twice with ice-cold PBS, scraped off and transferred to 1 ml cold active protease solution (PBS, 5 mg of Bacillus Licheniformis protease [Sigma, #P5380], 5 mM CaCl2, 20 U DNAse I [Roche, #4716728001]). Organoid tissue was incubated in a 2 ml reaction tube for 10-25 min on a slow moving shaker (nutator) in a coldroom at 4°C with repeated trituration steps for 10 seconds every 5 minutes. Single cell dissociation was confirmed with a hemocytometer under the microscope. The dissociation was stopped with 1 ml ice cold PBS supplemented with 10% fetal bovine serum (FBS). Afterwards the cells were immediately pelleted at 300x g for 5 min at 4°C. Subsequently, the supernatant was discarded and cells were suspended in 2 ml PBS/10%FBS and pelleted again at 300x g for 5 min at 4°C. Then cells were suspended in PBS/0.04%BSA and pelleted again (300x g for 5 min at 4°C), suspended in 1 ml PBS/0.04%BSA, and passed through a 30 µM filter mesh (Miltenyi MACS smart strainer). Viability was then investigated with the Trypan-blue exclusion test and cell concentration was determined with a hemocytometer and adjusted to 200,000 cells/ml for Drop-seq.Uniformly dispersed 1 nl-sized droplets were generated using self-built polydimethylsiloxane (PDMS) microfluidic co-flow devices on the basis of the AutoCAD design provided by the McCarroll group. The devices were treated with a water repellant solution (Aquapel) to create a hydrophobic channel surface. Drop-Seq runs followed closely the procedure published by Macosko et al. (Online Dropseq protocol v. 3.1 http://mccarrolllab.com/dropseq/). Barcoded beads (ChemGenes Corp., Wilmington, MA), suspended in lysis buffer, were co-flown with a single cell suspension and a droplet generation mineral oil (QX200, Bio-Rad Laboratories). Resulting droplets were collected in a 50 ml tube and immediately disrupted after adding 30 ml high-salt saline-sodium citrate buffer (6xSSC) and 1 ml perfluoro-octanol. Subsequently, captured mRNA’s were reverse transcribed for 2 hours using 2,000 U of the Maxima H Minus Reverse Transcriptase (ThermoFisher) followed by an exonuclease treatment for 45 minutes to remove unextended primers. After two washing steps with 6xSSC buffer about 70,000 remaining beads (60% of input beads) were aliquoted (5,000 beads per 50 µl reaction) and PCR-amplified (5 cycles at 65˚C and 12 cycles at 67˚C annealing temperature). Aliquots of each PCR reaction were pooled and double-purified using 0.5x volume of Agencourt AMPure XP beads (# A63881, Beckman Coulter) and finally eluted in 10 µl EB buffer. Quality and quantity of the amplified cDNAs were analyzed on a BioAnalyzer High Sensitivity DNA Chip (Agilent Technologies, Santa Clara, CA). About 600 pg cDNA was fragmented and amplified (17 cycles) to generate a next-generation sequencing library by using the Nextera XT DNA sample preparation kit (Illumina). |
