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Sample GSM2883192 Query DataSets for GSM2883192
Status Public on Jun 05, 2018
Title E15_5_wholeThy_1
Sample type SRA
 
Source name E15_5_wholeThy_1
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
Growth protocol Thymic lobes were isolated from timed mouse embryos.
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
 
Description Read 1 contains the barcode and Read 2 contains biological sequence
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.
 
Submission date Dec 11, 2017
Last update date Oct 11, 2019
Contact name Rene Maehr
Organization name UMass Medical School
Department Program in Molecular Medicine
Lab Diabetes Center of Excellence
Street address 55 Lake Ave N
City Worcester
State/province MA
ZIP/Postal code 01604
Country USA
 
Platform ID GPL19057
Series (1)
GSE107910 Single-cell RNA sequencing resolves cellular heterogeneity throughout embryonic development of the thymus
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 SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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