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Status |
Public on Jun 05, 2018 |
Title |
E15_5_wholeThy_1 |
Sample type |
SRA |
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Source name |
E15_5_wholeThy_1
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Organism |
Mus musculus |
Characteristics |
background strain: C57BL6/J genotype: wildtype tissue: thymus developmental day: E15.5 rbc lysis buffer: eBioscience Cat# 00-4333-57 treatment: NA
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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 |
Read 1 contains the barcode and Read 2 contains biological sequence
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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 T-Cell receptor (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. The FASTA with the TCR sequence is available on the series record. 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 |
Dec 11, 2017 |
Last update date |
Oct 11, 2019 |
Contact name |
Rene Maehr |
Organization name |
UMass Medical School
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Department |
Program in Molecular Medicine
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Lab |
Diabetes Center of Excellence
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Street address |
55 Lake Ave N
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City |
Worcester |
State/province |
MA |
ZIP/Postal code |
01604 |
Country |
USA |
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Platform ID |
GPL19057 |
Series (1) |
GSE107910 |
Single-cell RNA sequencing resolves cellular heterogeneity throughout embryonic development of the thymus |
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Relations |
Reanalyzed by |
GSE138691 |
BioSample |
SAMN08160115 |
SRA |
SRX3461260 |
Supplementary file |
Size |
Download |
File type/resource |
GSM2883192_E15_5_wholeThy_1.dge.txt.gz |
1.6 Mb |
(ftp)(http) |
TXT |
GSM2883192_E15_5_wholeThy_1_tcr.dge.txt.gz |
2.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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