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| Status |
Public on Apr 18, 2016 |
| Title |
Single_cell_RNA-seq_NKT1_36 |
| Sample type |
SRA |
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| Source name |
Single_cell_RNA-seq_NKT1
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
gender: female
age: 5 weeks
tissue: Thymus
cell type surface markers: CD24low, TCRßlow, NK1.1high, CD27high, CCR6−
vα14 inkt thymocyte subset: NKT1
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| Growth protocol |
NKT cells subtypes were isolated from Thymuses and directly sorted by Flow cytometry into a 96-well plate wells with lysis buffer with RNAase inhibitor (Takara) - Described in Picelli et al. Nat Protoc. 2014, PMID:24385147. No particular cell growth procedure was required.
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| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA isolation from thymic iNKT cell subsets, thymus cell suspensions were enriched for iNKT cells by negative selection of cells expressing CD8b.2, CD19 or TER-119 via magnetic selection utilizing reagents and equipment from Stem Cell Technologies. The remaining cells were then stained using an 11-parameter panel of reagents that included tetramers of CD1d loaded with either aGalcer or PBS57 combined with streptavidin-brilliant violet (BV)421 (produced in-house or obtained from the NIH Tetramer Core Facility), anti-CD44-V500 (BD Biosciences #56780), live/dead yellow (ThermoFisher Scientific #L34959), anti-CD4-Qdot 605 (ThermoFisher Scientific #Q10092), anti-CD103-FITC (Affymetrix #11-1031), anti-CD24-PerCPCy5.5 (Affymetrix #45-0242), anti-CD27-APC (Affymetrix #E07114), anti-CD8a-AF700 (Biolegend 100730), anti-TCRb-APC-eF780 (Affymetrix #47-5961), anti-CCR6-PE (Biolegend #129803), and anti-NK1.1-PE-Cy7 (BD Biosciences #552878). iNKT subsets were sorted using a FACSaria III (BD Biosciences).
Single cell RNAseq was perfromed as described in Picelli et al.Nat Protoc. 2014, PMID:24385147. We performed 22 cycles of amplification.
Single cell RNAseq was perfromed as described in Picelli et al.Nat Protoc. 2014, PMID:24385147. Barcoded Illumina sequencing libraries (Nextera XT library preparation kit, Illumina)
Whole Transcriptome Amplification (SmartSeq2 - Picelli et al.2014); Libraries were sequenced on the HiSeq2500 Illumina platform to obtain 50-bp single end reads (TruSeq® Rapid Kit, Illumina).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
SCRNA_NKT1_36
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| Data processing |
Single-cell RNA-Seq data was mapped against the mouse mm10 reference genome using tophat (v1.4.1., --library-type fr-secondstrand) and the RefSeq gene annotation downloaded from the UCSC genome Bioinformatics Site.
Genome_build: mm10.
Supplementary_files_format_and_content: abundance measurements
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| Submission date |
Nov 03, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Pandurangan Vijayanand |
| E-mail(s) |
vijay@lji.org
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| Organization name |
La Jolla Institute for Allergy and Immunology
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| Street address |
9420 Athena Circle
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE74596 |
Innate-like functions of natural killer T cell subsets result from highly divergent gene programs [single_cell_RNA-seq] |
| GSE74597 |
Innate-like functions of natural killer T cell subsets result from highly divergent gene programs |
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| Relations |
| BioSample |
SAMN04231015 |
| SRA |
SRX1410109 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1923525_SCT10_IsEn07_NKT1_N506_N704_W29_MappedReads_Annotations.txt.gz |
70.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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