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| Status |
Public on Feb 05, 2019 |
| Title |
NLW36b |
| Sample type |
SRA |
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| Source name |
Organoid
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| Organism |
Homo sapiens |
| Characteristics |
sample type: whole well extract
time point: Harvested day 19
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| Extracted molecule |
total RNA |
| Extraction 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).
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.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
paired end scRNASeq data
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| Data processing |
The quality of the fastq files from the sequencer were first checked using FastQC (v0.11.4).
Sequence aligned using Star
Next, using the tools embedded in Picard tools (picard-tools-1.115) and the DropSeq analysis pipeline developed by the McCarroll lab (http://mccarrolllab.com/dropseq/), the fastq files were processed and the data matrix table containing the gene expression of the barcoded cells was generated. Individual cells were labeled with barcodes, and transcripts within each cell were tagged with distinct UMIs (Unique Molecular Identifiers) in order to determine absolute transcript abundance.
Genome_build: GRCh38
Supplementary_files_format_and_content: Data matrix table is in text format
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| Submission date |
Jun 19, 2018 |
| Last update date |
Feb 05, 2019 |
| Contact name |
Rajasree Menon |
| E-mail(s) |
[email protected]
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| Phone |
7346159720
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| Organization name |
University of Michigan
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| Department |
Department of Computational Medicine and Bioinformatics
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| Street address |
4544E, MSRB2, Catherine Street, University of Michigan
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| City |
Ann Arbor |
| State/province |
Michigan |
| ZIP/Postal code |
48109 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE115986 |
Single-cell analysis of human kidney organoids |
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| Relations |
| BioSample |
SAMN09453297 |
| SRA |
SRX4235889 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3204321_NLW36b_GeneMatrix.txt.gz |
1.9 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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