|
| Status |
Public on May 13, 2020 |
| Title |
HUB-JB-033 |
| Sample type |
SRA |
| |
|
| Source name |
Human intestinal organoids
|
| Organism |
Homo sapiens |
| Characteristics |
genotype: Neurogenin 3 overexpression
tissue: Intestine
|
| Treatment protocol |
Live cells were sorted in 384-well hard shell plates (Biorad) with 5 μl of vapor-lock (QIAGEN) containing 100 nl of RT primers (consisting of a 24 bp polyT stretch, a 4bp random molecular barcode (UMI), a cell-specific barcode, the 5′ Illumina TruSeq small RNA kit adaptor and a T7 promoter), dNTPs and mRNA ERCC Spike-Ins (Agilent) and immediately frozen to −80°C. Before starting with SORT-seq, cells were first lysed 5 min at 65°C.
|
| Growth protocol |
Organoids were induced for Neurogenin 3 expression 5 days before sorting.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was processed using the previously described SORT-seq tecnique, which is an implemented version of the CEL-seq2 technique. Libraries were sequenced on an Illumina NextSeq500 using 60bp and 26bp paired end sequencing.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Cells were sorted into a 384 well plate and processed for singel cell sequencing
EEC_atlas_raw.csv
|
| Data processing |
Single cell data were demultiplexed, paired end reads were aligned to the transcriptome using bwa. Paired end reads obtained by CEL-seq were aligned to the transcriptome using bwa (version 0.6.2-r126) with default parameters. The transcriptome contained all RefSeq gene models based on the human genome release hg19. The right mate of each read pair was mapped to the ensemble of all gene loci and to the set of 92 ERCC spike-ins in sense direction. Reads mapping to multiple loci were discarded. The left read contains the barcode information: the first eight bases correspond to the cell specific barcode followed by 4 bases representing the unique molecular identifier. The remainder of the left read contains a polyT stretch followed by few (<15 transcript derived bases). The left read was not used for quantification. For each cell barcode we counted the number of unique molecular identifiers for every transcript and aggregated this number across all transcripts derived from the same gene locus.
Genome_build: hg19
Supplementary_files_format_and_content: The data are in csv format. The table contains UMI deduplicated, poisson corrected counts.
|
| |
|
| Submission date |
Apr 22, 2020 |
| Last update date |
May 13, 2020 |
| Contact name |
Jens Puschhof |
| E-mail(s) |
[email protected]
|
| Organization name |
Hubrecht Institute
|
| Street address |
Uppsalalaan 8
|
| City |
Utrecht |
| ZIP/Postal code |
3584CT |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE146799 |
Single cell and bulk RNA sequencing of Enteroendocrine cells from human hormone reporter organoids |
|
| Relations |
| BioSample |
SAMN14673630 |
| SRA |
SRX8158311 |
