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| Status |
Public on Jan 13, 2021 |
| Title |
Murine intestinal organoids - Dome-rep1 |
| Sample type |
SRA |
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| Source name |
Murine instestinal organoids
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| Organism |
Mus musculus |
| Characteristics |
tissue: Murine instestinal organoids
experiment: Sandwich v. Dome
culture type: Dome
treatment: untreated
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| Growth protocol |
Organoids were grown for 166 hours (one week) in ENR media prior to dissociation.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Organoids were grown in either sandwich or dome culture in glass-bottom 12 well plates (Mattek; P12G-1.5-14-F). Before dissociation, all pipette tips and transfer vessels were coated with 10% w/v BSA to prevent loss of material. Media was aspirated and culture wells were washed twice with 1X PBS before incubating in 1mL TrypLE Express (ThermoFisher Cat No. 12604-021). Upon addition of TrypLE Express, a 1 mL pipette tip was used to mechanically detach organoids embedded in Matrigel from either sandwich or dome cultures, taking care to scrape the bottom of the well with such force so as not to detach the glass bottom coverslip, but to visibly remove the Matrigel from the coverslip. Organoids were incubated at 37°C for between 20-30 minutes, monitoring every 5 minutes to check on the status of dissociation, and to repeat brief mechanical disruption by trituration. Multiple wells (between 2 and 10) per condition were pooled into a 15 mL falcon tube and diluted 1:1 with ice cold DMEM, and strained (40 μm [pluriSelect 43-10040-60]) into a 15 mL falcon tube. This cell solution was then centrifuged at 250xg in a spinning bucket centrifuge for 1 minute at 4°C, and the suspension was aspirated. Cells were resuspended in 200 μL of 1% w/v BSA in 1X PBS, kept on ice, and cell count and viability were measured with a hemocytometer immediately prior to single cell encapsulation with the inDrops method.
InDrops single cell RNA sequencing was performed as described before [Zilionis et al., 2019, Immunity, 50, 1317-1334.e10; Zilionis et al., 2017, Nat Protoc, 12, 44-73; Klein et al., 2015, Cell, 161, 1187-1201] with changes made as written in the publication associated with this GEO record. All reads were processed with the indrops.py pipeline found at https://github.com/AllonKleinLab/klunctions/tree/master/sam/inDrops_pipeline using the parameters included in the provided bash script files: submit_indrops_sandwich_dome_rc.sh and submit_indrops_perturb2.sh, for sandwich v. dome and perturbation experiments, respectively.
scRNA-seq
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
adata_mg_annotated_merged_coarse_grain_no_ss_12092020.h5ad
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| Data processing |
Basecalling to obtain fastq files as described in https://github.com/AllonKleinLab/klunctions/tree/master/sam/inDrops_pipeline using the supplementary bash scripts bcl2fastq.sh and bcl2fastq_perturb2.sh for each of the dome v. sandwich and perturbation experiments, respectively.
Gene expression counts in single cells were obtained from raw fastq files using the indrop.py pipeline from https://github.com/AllonKleinLab/klunctions/tree/master/sam/inDrops_pipeline on 2020 07 22 and 2020 09 04; parameters are listed in supplementary yaml files: 20200722_sandwich_dome_rc.yaml and 20200904_perturb_rc.yaml for the dome v. sandwich and perturbation experiments, respectively.
The processed data files in for each sample consists of an annotated tsv.gz file with raw unfiltered gene counts (cells x genes), gene names, and barcodes; this file can be parsed into its respective matrix, barcodes, and genes using the following python command: hf.load_annotated_text(
hf.file_opener(input_path + s + '.counts.tsv.gz'),
delim='\t',
read_row_labels=True,
read_column_labels=True)
Genome_build: mm10; supplementary fasta formatted bowtie index with GFP sequences: mouse.GRCm38_withGFP.idx.fa
Supplementary_files_format_and_content: The processed data files in for each sample consists of an annotated tsv.gz file with raw unfiltered gene counts (cells x genes), gene names, and barcodes; this file can be parsed into its respective matrix, barcodes, and genes using the python command described above.
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| Submission date |
Jan 12, 2021 |
| Last update date |
Jan 13, 2021 |
| Contact name |
Allon M Klein |
| E-mail(s) |
Allon_Klein@hms.harvard.edu
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| Organization name |
Harvard Medical School
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| Department |
Systems Biology
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| Street address |
210 Longwood Avenue
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE164638 |
Inflation-collapse dynamics drive patterning and morphogenesis in intestinal organoids |
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| Relations |
| BioSample |
SAMN17293620 |
| SRA |
SRX9832152 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5015811_Dome-R1-2.1-filtered.h5ad.gz |
4.2 Mb |
(ftp)(http) |
H5AD |
| GSM5015811_Dome-R1-2.1-raw.h5ad.gz |
6.3 Mb |
(ftp)(http) |
H5AD |
| GSM5015811_Dome-R1-2.1.abundant_barcodes.csv.gz |
98.2 Kb |
(ftp)(http) |
CSV |
| GSM5015811_Dome-R1-2.1.counts.tsv.gz |
7.1 Mb |
(ftp)(http) |
TSV |
| GSM5015811_Dome-R1-2.1.genes.txt.gz |
102.4 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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