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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jun 01, 2020 |
| Title |
week6_preselected_gd_1 |
| Sample type |
SRA |
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| Source name |
library of 96 WT preselected gamma delta T cells
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| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL/6
genotype/variation: wild type
Sex: female
age/developemental stage: 5-6 weeks
tissue: Thymus
cell type: preselected gamma delta T cells
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| Treatment protocol |
Maf KO, Sox13 Ko and Rorc KO cells were kept frozen in 90 % fetal bovine serum (FBS) and 10 % Dimethyl sulfoxide (DMSO) -80ºC before performing antibody staining, FACS sorting and single cell RNA sequencing.
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| Growth protocol |
WT mice were kept in the animal facility of the Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany. Maf KO mice were kept in the animal facility of The Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, New York, USA. Sox13 KO mice were kept in the animal facility of Institut für Biochemie, Emil-Fischer-Zentrum, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany. Rorc KO mice were kept in the animal facility of Institute of Medical Microbiology and Hygiene, University Medical Center Freiburg, Freiburg, Germany.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Thymocytes were isolated using mechanical dissociation of thymus. Since double negative (DN) thymocytes are rare, we performed the magnetic enrichment for DN thymocytes.
As described in mCEL-Seq2 protocol (Hashimshony et al. 2016 and Herman et al. 2018)
Adapted from TruSeq Small RNA Library Preparation Protocol
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
library of 96 WT preselected gamma delta T cells
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| Data processing |
For image aquisition, intensity extraction and basecalling HiSeq Control Software 2.0.2, RTA 2.4.11 / Recipe Fragment 2.0.0.2 was used. Conversion of bcl2fastq files was performed using bcl2fastq 2.17.1.14
Paired end reads were aligned to the transcriptome using bwa (version 0.6.2-r126) with default parameters. The transcriptome contained all gene models based on the mouse ENCODE VM9 release downloaded from the UCSC genome browser comprising 57,207 isoforms derived from 57,207 gene loci with 57,114 isoforms mapping to fully annotated chromosomes (1 to 19, X, Y, M). All isoforms of the same gene were merged to a single gene locus. Furthermore, gene loci overlapping by >75% were merged to larger gene groups. This procedure resulted in 34,111 gene groups.
The right mate of each read pair was mapped to the ensemble of all gene loci and to the set of 92 ERCC spike-ins in sense direction. Reads mapping to multiple loci were discarded. The left read contains the barcode information: the first six bases correspond to the cell specific barcode followed by six bases representing the unique molecular identifier (UMI). The remainder of the left read contains a poly(T) stretch. The left read was not used for quantification. For each cell barcode, the number of UMIs per transcript was counted and aggregated across all transcripts derived from the same gene locus.
Based on binomial statistics, the number of observed UMIs was converted into transcript counts (Gruen et al., 2014).
Genome_build: ENCODE VM9
Supplementary_files_format_and_content: CSV files, columns represent each cell barcode (total barcodes used = 192), rows represent the geneid and the values in the file are the quantified number of transcripts.
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| Submission date |
Jun 13, 2018 |
| Last update date |
Jun 01, 2020 |
| Contact name |
Sagar - |
| E-mail(s) |
sagar@uniklinik-freiburg.de
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| Organization name |
University Medical Center Freiburg
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| Department |
Department of Internal Medicine II
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| Lab |
Sagar
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| Street address |
Hugstetter Straße 55
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| City |
Freiburg |
| ZIP/Postal code |
79106 |
| Country |
Germany |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE115765 |
Deciphering the regulatory landscape of fetal and adult gd T-cell development at single-cell resolution |
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| Relations |
| BioSample |
SAMN09407128 |
| SRA |
SRX4211815 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3188738_week6_preselected_gd_1.coutt.csv.gz |
554.7 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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