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Sample GSM2464256 Query DataSets for GSM2464256
Status Public on Dec 24, 2019
Title RNA_CLP_1
Sample type SRA
 
Source name common lymphoid progenitor (CLP)
Organism Mus musculus
Characteristics strain: C57BL/6
cell type: common lymphoid progenitor (CLP)
tissue: isolated from bone marrow
Extracted molecule polyA RNA
Extraction protocol Single cell suspension were obtained from the bone marrow of C57BL/6 mice. For sorting CLP and rChILP, the lineage negative cells were enriched with the "Lineage Cell Depletion Kit, mouse" from Miltenyi Biotec according to the manual. Enriched Lineage negative bone marrow cells were stained with relative antibodies followed by sorting on a cell sorter FACS Aria III (BD Biosciences) for "live Lineage-CD127+Flt-3+Integrin α4β7-Sca1+c-Kit+" CLP and "live Lineage-CD127+Flt-3-Integrin α4β7+T1/ST2-c-Κit+" rChILP. For sorting PD-1+ non-LTi ILC progenitors and CCR6+ LTi progenitors, the Fc receptor-blocked bone marrow single-cell suspension was stained with PE-conjugated anti-integrin α4β7 antibody, followed by washing and incubation with anti-PE microbeads. The samples were then applied to the Miltenyi LS columns in magnetic field and washed three times by PBS with 3% FBS according to the manufacturer’s instruction. The integrin α4β7+ cells on the column were finally flushed into a collection tube for further surface molecule staining. Non-LTi ILC progenitors (live Lineage-CD127+Flt-3-Integrin α4β7+T1/ST2-c-Κit+PD-1+CCR6-) and LTi progenitors (live Lineage-CD127+Flt-3-Integrin α4β7+T1/ST2-c-Κit+PD-1-CCR6+) were then sorted via FACS Aria III (BD Biosciences). CLPs, total rChILPs, PD-1+ or CCR6+ progenitors described above were sorted directly into 700 μL of QIAzol Lysis Reagent in the miRNAeasy Micro Kit (QIAGEN, Cat#217084). Total RNA was extracted and RNA-Seq libraries were prepared through the Smart-seq2 method (PMID: 24385147) as previously described (PMID: 29466755). Multiplex sequencing reads of 50 bp were generated by the NHLBI DNA Sequencing and Computational Biology Core.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 3000
 
Data processing Basecalls performed using CASAVA 1.8.4
Sequence reads were mapped to the mouse (mm9) genomes with bowtie2 (2.2.2) with default settings. We kept one hit if multiple reads were mapped to the same position for each RNA-Seq libraries.
For each RNA-Seq sample, gene expression was quantified by RPKM with in-house script and RefSeq gene annotation.
Genome_build: mm9
Supplementary_files_format_and_content: rpkm: plan text file. Columns from left to right denote RefSeq ID, RPKM, gene transcript size, number of read mapped to exons, a combination of RefSeq ID, gene symbol, etc, and reserved column for future development.
 
Submission date Jan 19, 2017
Last update date Dec 24, 2019
Contact name Gangqing Hu
E-mail(s) michael.hu@hsc.wvu.edu
Organization name West Virginia University
Department MicroBiology, Immunology, and Cell Biology
Lab 2072A, HSC North, Floor 2
Street address 64 Medical Center Drive
City Morgantown
State/province West Virginia
ZIP/Postal code 26506-9177
Country USA
 
Platform ID GPL21493
Series (1)
GSE93863 Differential Expression of the Transcription Factor GATA3 Specifies Lineage and Functions of Innate Lymphoid Cells
Relations
BioSample SAMN06242763
SRA SRX2505749

Supplementary file Size Download File type/resource
GSM2464256_GA9332-CGATGT-RNA-seq-mouse-CLP_0_0.rpkm.gz 481.7 Kb (ftp)(http) RPKM
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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