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Status |
Public on Feb 15, 2019 |
Title |
Rag2null_E14_5_WholeThy_rep1 |
Sample type |
SRA |
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Source name |
Rag2null_E14_5_WholeThy_rep1
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Organism |
Mus musculus |
Characteristics |
genotype: B6(Cg)-Rag2tm1.1Cgn/J source tissue: embryo developmental day: E14.5 rbc lysis buffer: none cell type: mixed sample (non-demultiplexed) [whole thymus, including mesenchyme, endothelium, epithelium, thymocytes, and other lymphocytes]
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Growth protocol |
Thymic lobes were isolated from timed mouse embryos.
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Extracted molecule |
polyA RNA |
Extraction protocol |
Thymic lobes were mechanically isolated, dissociated in trypsin, depleted of RBC's (only some samples), washed and stained with 7AAD cell viability dye, and 7AAD negative single cells were sorted on a BD FACSARIA. Libraries were prepared according to the Drop-seq lab protocol version 3.1 (Macosko and Goldman, December 2015, McCarroll Lab). Final libraries were sequenced on an Illumina NextSeq 500 using high-output 75-cycle kits.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NextSeq 500 |
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Description |
Drop-seq
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Data processing |
Base calling was performed using Illumina RTA version 2.4.11. FASTQ's from resequenced runs were concatenated prior to processing. Alignment and quantification were performed using the STAR aligner v2.4.2, Picard tools v1.96, Samtools v1.3, and Drop-seq tools v1.0. Analysis step: Convert to SAM Software package: Picard tools Command name: FastqToSam.jar Parameters used: -- Analysis step: Tag cell barcode Software package: DropSeq tools Command name: TagBamWithReadSequenceExtended Parameters used: BASE_QUALITY=10, BARCODED_READ=1, NUM_BASES_BELOW_QUALITY=1, DISCARD_READ=False Analysis step: Tag molecular barcode Software package: DropSeq tools Command name: TagBamWithReadSequenceExtended Parameters used: BASE_QUALITY=10, BARCODED_READ=1, NUM_BASES_BELOW_QUALITY=1, DISCARD_READ=True Analysis step: Remove low-quality barcodes Software package: DropSeq tools Command name: FilterBAM Parameters used: -- Analysis step: Remove polyA tail Software package: DropSeq tools Command name: PolyATrimmer Parameters used: MISMATCHES=0, NUM_BASES=6 Analysis step: Convert to FASTQ Software package: Picard tools Command name: SamToFastq.jar Parameters used: -- Analysis step: Align reads to exome Software package: STAR Command name: -- Parameters used: -- Analysis step: Sort BAM to speed merging Software package: Picard tools Command name: SortSam.jar Parameters used: -- Analysis step: Merge barcode and alignment info Software package: Picard tools Command name: MergeBamAlignment.jar Parameters used: INCLUDE_SECONDARY_ALIGNMENTS=False Analysis step: Label read with exon Software package: DropSeq tools Command name: TagReadWithGeneExon Parameters used: -- Analysis step: Screen for bead errors Software package: DropSeq tools Command name: DetectBeadSynthesisErrors Parameters used: NUM_BARCODES=2000 Analysis step: Form DGE matrix Software package: DropSeq tools Command name: DigitalExpression Parameters used: MIN_NUM_GENES_PER_CELL=1000 The procedure was repeated with a TCR contig added to the reference genome and the following two steps were used to generate improved TCR counts. Analysis step: Subset reads aligning to TCR contig Software package: Samtools Command name: view Parameters used: -bh Analysis step: Form TCR-specific DGE matrix Software package: DropSeq tools Command name: DigitalExpression Parameters used: MIN_NUM_GENES_PER_CELL=1 READ_MQ=1 Genome_build: For initial DGE calculations, mm10 was used. For TCR-specific DGE's, an artificial TCR contig was added. Supplementary_files_format_and_content: Processed files are digital gene expression matrices containing UMI counts. They are represented as dense matrices in (gzipped) tab-delimited text files. Each column is labeled with a cell barcode and each row is labeled with a gene.
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Submission date |
Feb 14, 2019 |
Last update date |
Feb 15, 2019 |
Contact name |
David Sebastian Fischer |
Organization name |
Helmholtz Zentrum Munich
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Department |
ICB
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Lab |
Theis
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Street address |
Ingolstaedter Landstr. 1
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City |
Neuherberg |
ZIP/Postal code |
85764 |
Country |
Germany |
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Platform ID |
GPL19057 |
Series (1) |
GSE126579 |
Inferring population dynamics from single-cell RNA-sequencing time-series data |
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Relations |
BioSample |
SAMN10939291 |
SRA |
SRX5379467 |
Supplementary file |
Size |
Download |
File type/resource |
GSM3608106_Rag2null_E14_5_WholeThy_rep1_star_merged_gene_exon_tagged_clean.dge.txt.gz |
1.4 Mb |
(ftp)(http) |
TXT |
GSM3608106_star_Rag2null_E14_5_WholeThy_rep1_TCR.dge.txt.gz |
650 b |
(ftp)(http) |
TXT |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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