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|Public on Nov 10, 2021
|culture method: Ex vivo cell culture
sample day: SampleDay0
tissue: Intestinal organoid
|After matrigel polymerization, cells were cultured in DMEM/F12 supplemented with Glutamine, Hepes, penicillin-streptomycin, B27 supplement, N2, and N-Acetyl-L-cysteine, growth factors R-spondin, mNoggin, EGF (called ENR), Y-27632 p160ROCK inhibitor. To promote cell proliferation CHIR and Valproic acid (collectively called CV) were added three days post embedding. Medium was replenished after 2 days of culture. After three days of proliferation, medium was changed to a differentiation growth medium (ENR only). Organoid sampling performed three days after culturing in ENR CV condition, and during three days of differentiation in ENR.
|Intestinal organoids used were isolated from mouse crypts from dissociated mouse intestinal stem cell colonies from 5-10 week old heterozygous Lgr5-eGFP-IRES-CreERT2 mice. Isolation of Lgr5 eGFP+ stem cell population and initial culture was performed as described elsewhere. For the developmental time-course experiments, organoids were dissociated to single-cells, live Lgr5+-eGFP cells isolated using a FACS ARIA II (BD) and embedded in Matrigel. After matrigel polymerization, cells were cultured in DMEM/F12 supplemented with Glutamine, Hepes, penicillin-streptomycin, B27 supplement, N2, and N-Acetyl-L-cysteine, growth factors R-spondin, mNoggin, EGF (called ENR), Y-27632 p160ROCK inhibitor. To promote cell proliferation CHIR and Valproic acid were added three days post embedding (collectively called CV). Medium was replenished after 2 days of culture. After three days of proliferation, medium was changed to a differentiation growth medium (ENR only). Organoid sampling performed three days after culturing in ENRCV condition, and during three days of differentiation in ENR.
|Single organoids collected by dissolving Matrigel with cold Cell Recovery Solution, followed by hand-picking and transferring to Nunc microwell culture plate with a dissociation mix containing protease isolated from B. licheniformis, EDTA, EGTA, DNase I, Accutase and PBS. Single organoids were dissociated by combining trituration using a siliconized pipette tips every 5 minutes and incubation at 37oC on for 15 minutes total. Following the dissociation, cell suspensions were diluted cell loading buffer in the loading tip of the DisCo chip.
After bead-cell in droplet co-encapsulation, the gel loading tip containing the sample droplets was transferred to a bead collection chip inlet (cp-chip; as described in Lab on a chip). Droplets in the tip were flushed to bead collection chip. Subsequently to bead capture, washing was performed as in Drop-seq with SSC and Reverse transcription buffer directly in the recovery chip. Reverse transcription solution was added to the beads and the recovery chip was placed on a heating block to perform first strand cDNA synthesis (RT) for 90 minutes at 42oC. After RT reaction beads were washed on the recovery chip with TE-SDS once, with TE-TW twice and with Tris once. The beads were treated with Exonuclease I for 45 minutes at 37oC to remove single-stranded oligonucleotides on beads. After Exonuclease I treatment, beads were washed with TE-SDS once, with TE-TW twice (as after reverse transcription). Beads were then eluted from the recovery chip in dH2O. cDNA was amplified for 18 – 23 cycles using Kapa HiFi Hot start ready mix. cDNA was purified with CleanPCR magnetic beads using 0.6X ratio to remove small cDNA fragments and primers. To assess the cDNA yield and quality concentration was measured using Qubit and cDNA traces quality was assessed using Fragment Analyzer (Agilent). cDNA was tagmented with in-house Tn5 (generated as described in Picelli et al..) and the library was purified using CleanPCR magnetic beads (0.6X ratio).
Libraries were sequenced on a NextSeq 500 system (Illumina) following recommendations from original protocol (20 bp for read 1 and 50 bp for read2).
|Illumina NextSeq 500
|Drop-seq tools 2.3.0 package on the EPFL SCITAS HPC platform. After trimming and sequence tagging, reads were aligned to the mouse reference genome (mm10) using STAR (version 2.7.0.e). https://github.com/mccrowjp/Dropseq
Following the alignment, the gene annotation was added, bead synthesis errors were corrected and cell barcodes extracted. Subsequently, the BAM files containing the processed data were used to obtain digital gene expression matrices.
Downstream data analysis was carried out usingR (version 3.5.0) using Seurat (version 3.1.1) and uwot (version 0.1.3).
Supplementary_files_format_and_content: tab-delimited text files include read-count matrices for each organoid, Seurat-Object
|Apr 05, 2020
|Last update date
|Nov 10, 2021
|Laboratory for systems biology and genetics/UPDE
|Station 19, SV 3820
|Low-input, deterministic profiling of single-cell transcriptomes reveals individual intestinal organoid subtypes comprised of single, dominant cell types [organoids]
|Low-input, deterministic profiling of single-cell transcriptomes reveals individual intestinal organoid subtypes comprised of single, dominant cell types