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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Nov 10, 2021 |
| Title |
Bulke4 |
| Sample type |
SRA |
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| Source name |
Intestinal crypts
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| Organism |
Mus musculus |
| Characteristics |
sample type: Ex vivo
tissue: several crypts
batch: batch5
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| Growth protocol |
Intestinal crypts used were isolated from mouse small intestine. Single crypts were hand-picked and dissociated to cell suspensions. Cell suspensions for bulk samples were obtained from a random sample of non-handpicked crypts.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
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 to in Lab on a chip). Droplets in the tip were flushed to bead collection chip. After beads were captured, washing was performed as in Drop-seq with 6X SSC and Reverse transcription buffer using the same tip. Reverse transcription solution was added to beads and recovery chip was placed on a heating block.
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).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Drop-seq tools 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).
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include read-count matrices for each organoid
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| Submission date |
Sep 08, 2021 |
| Last update date |
Nov 10, 2021 |
| Contact name |
Marjan Biočanin |
| E-mail(s) |
marjan.biocanin@epfl.ch
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| Organization name |
EPFL
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| Department |
SV-IBI
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| Lab |
Laboratory for systems biology and genetics/UPDE
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| Street address |
Station 19, SV 3820
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| City |
Lausanne |
| State/province |
Vaud |
| ZIP/Postal code |
1015 |
| Country |
Switzerland |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE148093 |
Low-input, deterministic profiling of single-cell transcriptomes reveals individual intestinal organoid subtypes comprised of single, dominant cell types |
| GSE183691 |
Low-input, deterministic profiling of single-cell transcriptomes reveals individual intestinal organoid subtypes comprised of single, dominant cell types [Intestinal crypts] |
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| Relations |
| BioSample |
SAMN21357895 |
| SRA |
SRX12105496 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5567823_Bulk_Crypt10_S6.txt.gz |
275.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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