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| Status |
Public on Nov 09, 2016 |
| Title |
Untreated-Ctrl-03182015_S3 |
| Sample type |
SRA |
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| Source name |
Pancreatic Islet
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| Organism |
Homo sapiens |
| Characteristics |
bulk sample type: Intact
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 |
Bulk cells were pelleted and RNA purified using the PicoPure RNA isolation kit (Life Technology).
All RNA-seq libraries from bulk sample RNA were generated with the same SMARTer v1 chemistry (Clontech) as the C1 single cell data largely following manufacturers instructions. Unlike the C1 workflow, after first strand DNA synthesis cDNA was purified using Agencourt AMPure beads (Beckman Coulter). cDNA was subsequently amplified through 12 PCR cycles. The cDNA yield and fragment size was measured on a 2100 Bioanalyzer (Agilent). For sequencing library preparation, amplified cDNA was sheared using a Covaris LE220 system to obtain fragments of approximately 200 bp. The fragmented cDNA was prepared for sequencing using the NEBNext DNA Library Prep Kit for Illumina sequencing (New England Biolabs).
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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.
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. There are 24 total bulk islet samples in this processed file.
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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) |
| GSE86468 |
Single cell transcriptomics defines human islet cell signatures and reveals cell-type-specific expression changes in type 2 diabetes [bulk] |
| 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 |
SRX1813631 |
| BioSample |
SAMN05190615 |
| 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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