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Status |
Public on Jun 03, 2018 |
Title |
WT [June29_KW.38.featurecounts] |
Sample type |
SRA |
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Source name |
Thymus epithelium
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Organism |
Mus musculus |
Characteristics |
strain: C57BL/6.Il25 genotype: Flare25/Flare25 age: 6 weeks Sex: Mixed mouse status: Naive sorting gate: UVBlue-;CD11c-;CD45-;EPCAM+;MHCIIlo;Flare25+
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Extracted molecule |
polyA RNA |
Extraction protocol |
For thymic epithelium, thymi were isolated, cleaned of fat and transferred to DMEM + 2% FBS on ice. Thymi were minced with a razor blade and tissue pieces were moved with a glass Pasteur pipette to 15 ml tubes and vortexed briefly in 10 ml of media. Fragments were allowed to settle before removing the media and replacing it with 4 ml of digestion media containing 2% FBS, 100 μg mL-1 DNase I (Roche), and 100 μg mL-1 Liberase TM (Sigma-Aldrich) in DMEM. Tubes were moved to a 37° C water bath and fragments were triturated through a glass Pasteur pipette at 0 min and 6 min to mechanically aid digestion. At 12 min, tubes were spun briefly to pellet undigested fragments and the supernatant was moved to 20 ml of 0.5% BSA (Sigma-Aldrich), 2 mM EDTA (TekNova), in PBS (MACS buffer) on ice to stop the enzymatic digestion. This was repeated twice for a total of three 12 min digestion cycles, or until there were no remaining tissue fragments. The single cell suspension was then pelleted and washed once in MACS Buffer. Density-gradient centrifugation using a three-layer Percoll gradient (GE Healthcare) with specific gravities of 1.115, 1.065, and 1.0 was used to enrich for stromal cells. Cells isolated from the Percoll- light fraction, between the 1.065 and 1.0 layers, were then washed and resuspended for staining. Cells were stained with fluorescently labeled antibodies and sorted via flow cytometry into single wells of 96 well plates containg 5 ul of lysis buffer. cDNA libraries were prepared using the standard smartSeq-2 protocol including ERCC control RNA. RNA libraries were prepared for sequencing using standard Illumina protocols
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2500 |
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Description |
June29_KW.38.featurecounts Corresponds with batch 1 of methods POR_all_data.txt.gz all_wt_counts.txt.gz POR_all_data.rda
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Data processing |
Reads were aligned to the GRCm38 build of the Mus musculus genome using STAR (release 020201) with standard parameters. Initial cell filtering was performed by setting a minimum threshold of 40% total reads mapping to the genome. Read counting to genes was performed with featureCounts (version 1.5.3) from the Subread package. Non-default parameters used were --primary. Gene filtering was performed by setting a minimum threshold of 10 reads in a total of 5 cells. Cell filtering was performed by requiring a minimum of 750 genes expressed with 10 reads. Futhermore, we required cells to have a minimum of 100,000 total reads mapping to known genes and less than 10% of reads mapping to mitochondrial genes. This retained 197 WT cells and 95 AireKO cells for downstream analysis. Normalization was performed using the indeXplorer pipeline (https://git.embl.de/velten/indeXplorer) (version ad03ee7f), depending on SCDE version 2.6.0. Normalized files are provided. Mitochondrial read count effect was regressed out of the data. All downstream analysis was performed using R version 3.4.0. All analysis code and pipelines are provided at the publically available repository https://github.com/mTEC-pipelines/tufts-pipelines. Genome_build: GRCm38 Supplementary_files_format_and_content: Expression matriix of post-normalized read counts for all retained cells. Supplementary_files_format_and_content: aireko_counts.txt: tab-separated file of raw counts, aireKO samples Supplementary_files_format_and_content: all_wt_counts.txt: tab-separated file of raw counts, wild-type samples Supplementary_files_format_and_content: POR_all_data.txt: tab-separated file of normalized expression counts Supplementary_files_format_and_content: samples_all.txt: tab-separated metadata file with sample batch information Supplementary_files_format_and_content: POR_all_data.rda: Rdata file of normalized expression counts
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Submission date |
May 24, 2018 |
Last update date |
Jun 03, 2018 |
Contact name |
Aparna Rajiv Rajpurkar |
E-mail(s) |
arrajpur@stanford.edu
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Organization name |
Stanford University
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Department |
Genetics
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Lab |
Steinmetz
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Street address |
279 Campus Drive
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City |
Stanford |
State/province |
CA |
ZIP/Postal code |
94305 |
Country |
USA |
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Platform ID |
GPL17021 |
Series (1) |
GSE114895 |
Thymic tuft cell single-cell mRNA analysis |
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Relations |
BioSample |
SAMN09255809 |
SRA |
SRX4122519 |
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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