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| Status |
Public on Nov 09, 2016 |
| Title |
1st-C15_S3 |
| Sample type |
SRA |
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| Source name |
Pancreatic Islet
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Alpha
tissue: Pancreatic Islet
Sex: Male
disease: Non-Diabetic
age: 22
race: African American
bmi: 32.95
islet unos id: ACCG268
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| Growth protocol |
Islet Acquisition, Processing, and Dissociation. Islets have been acquired through IIDP and shipped in Prodo-media overnight on cold packs, washed and taken into 37oC 5% CO2 Prodo-media culture immediately after arrival. Twenty-four hours after taken into culture an adequate volume of islets (500IEq) was aliquoted and centrifuged at 180xg 3min @RT. The first aliquot (100IEq) was straight flash frozen (= baseline), the second aliquot (200IEq) was resuspend in 1ml Prodo-media (= bulk) and the third aliquot (200IEq) resuspend in 1ml Accutase (Innovative Cell Technologies, Inc.) (= single cell) and incubated for 10min @37oC water bath, with pipetting every 2min. The second aliquot in Prodo-media was divided (100IEq each) and kept one @RT and one on ice. While the Accutase sample was put through pre-wet cell strainer (BD) and rinsed with 9ml pre-warmed CMRL+10%FBS media to stop reaction and centrifuged at 180xg 3min @RT. Dissociated cells were measured in 300ul CMRL+10%FBS media on a Countess II FL (Thermo Fisher Scientific) to assess cell number and viability and diluted down to 300 cells/ul. Total processing and handling time for each islet was ≤60 minutes.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Single Cell Processing on the C1 Single Cell Autoprep System. After enumeration, cells were diluted to a final concentration range of 250–400 cells per μl and 5 ul loaded onto each C1 integrated fluidic circuit (IFC; 10- to 17-μm chip) for cell capture on the C1 Single-cell Auto Prep System.
After capture cells were imaged within each capture nest with an EVOS FL Auto microscope (Life Technologies). IFCs were subsequently loaded with additional reagents for subsequent cell lysis, and SMARTer v1-based (Clontech), olio-(dT)-primed reverse transcription, template switching for second strand priming, and amplification of cDNA on the C1 System. Qualitative and quantitative analysis of all single cell cDNA products was performed on a 96 capillary Fragment Analyzer (Advanced Analytical). Only cell singlets, as determined by imaging, with adequate cDNA yield and quality were processed for subsequent sequencing. Fragmentation and tagmentation of cDNA was done with Nextera XT regarent (Illumina) using dual indices to prepare single-cell multiplexed libraries.
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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 |
Sequencing, Read Mapping, and Quality Control. All sequencing was performed on a NextSeq500 (Illumina) using the 75 cycle high output chip. RNA-seq reads were subjected to quality control using custom scripts developed at the computational sciences group at The Jackson Laboratory. Briefly, reads with more than 30% of bases with quality scores were removed from the analysis and samples with more than 50% reads removed are removed from further analysis. Trimmed reads were mapped to human transcriptome (GRCh37, ensemble v70) using Bowtie2 (Langmead and Salzberg, 2012) and expression levels of all genes were estimated using RSEM (Li and Dewey, 2011). Transcript per million (TPM) values as defined by RSEM were added a value of 1 (to avoid zeros) prior to log2 transformation.
Single Cell Sample Processing and Quality Filtering. 26,616 protein-coding genes and long non-coding RNAs (lincRNAs) from the GRCh37, ensemble v70 build were used in our study. Genes with expression levels greater than or equal to 5 TPM in a sample were considered to be expressed. 72 single cell samples that expressed fewer than 3500 genes according to these criteria were removed from downstream analysis. Approximately one-third (340/978, or 35%) of cells expressed more than one marker gene; these were removed from subsequent analysis owing to concerns that these were the result of two vertically stacked cells in a given capture site. Ultimately, there are 638 samples comprising this processed dataset.
Islet Cell Type Classification. In general, density plots of marker gene expression in our samples portray a bimodal distribution. Therefore, Gaussian mixture modeling was fitted on a per gene basis using two mixture components to identify in which samples each gene was expressed. This Gaussian mixture modeling was performed using the R-package, mclust_5.2 (Fraley et al. 2016). Each single cell sample was classified as a specific pancreatic cell type if and only if a single gene from the selected marker gene list: INS (beta), GCG (alpha), SST (delta), PPY (gamma/PP), KRT19 (ductal), PRSS1 (acinar), and COL1A1 (stellate) was expressed in the sample and none of the other marker genes were expressed. If more than one of these marker genes were expressed in a single cell, it was given the label “multiple”. If none of the marker genes were expressed it was labeled as “none”.
Genome_build: GRCh37, ensembl v70
Supplementary_files_format_and_content: Comma-separated value file contains raw transcript per million (TPM) values of 26,616 protein coding and long non-coding RNAs. The files are matrices with rows representing features (genes) and columns representing samples (638 total).
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| Submission date |
Sep 06, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Michael Stitzel |
| E-mail(s) |
Michael.Stitzel@jax.org
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| Organization name |
The Jackson Laboratory
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| Street address |
10 Discovery Drive
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| City |
Farmington |
| State/province |
CT |
| ZIP/Postal code |
06032 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE86469 |
Single cell transcriptomics defines human islet cell signatures and reveals cell-type-specific expression changes in type 2 diabetes [single cell] |
| GSE86473 |
Single cell transcriptomics defines human islet cell signatures and reveals cell-type-specific expression changes in type 2 diabetes |
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| Relations |
| SRA |
SRX1813994 |
| BioSample |
SAMN05190978 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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