Growth Protocol:	-
Treatment Protocol:	-
Extract Protocol:	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).
Library Construction Protocol:	The libraries were purified, quantified (Agilent High sensitivity DNA chip), and then sequenced (paired end 26x115 bases) on an Illumina HiSeq2500 platform (Rapid mode). Custom primer (5’-GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC-3’) was used for the first sequence read to identify all different cell barcodes und unique molecular identifier (UMI) sequences.

Molecule Type:	poly(A)+ RNA
Library Source:
Library Layout:	PAIRED
Library Strand:	Forward
Platform:	ILLUMINA
Instrument Model:	Illumina HiSeq 2000
Strand-Specific:	Specific

Data Resource	GEN Sample ID	GEN Dataset ID	Project ID	BioProject ID	Sample ID	Sample Name	BioSample ID	Sample Accession	Experiment Accession	Release Date	Submission Date	Update Date	Species	Race	Ethnicity	Age	Age Unit	Gender	Source Name	Tissue	Cell Type	Cell Subtype	Cell Line	Disease	Disease State	Development Stage	Mutation	Phenotype	Case Detail	Control Detail	Growth Protocol	Treatment Protocol	Extract Protocol	Library Construction Protocol	Molecule Type	Library Layout	Strand-Specific	Library Strand	Spike-In	Strategy	Platform	Instrument Model	Cell Number	Reads Number	Gbases	AvgSpotLen1	AvgSpotLen2	Uniq Mapping Rate	Multiple Mapping Rate	Coverage Rate