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Status |
Public on May 21, 2019 |
Title |
Human fetal liver (D7_122#658) |
Sample type |
SRA |
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Source name |
Human fetal liver
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Organism |
Homo sapiens |
Characteristics |
sample type: Freshly isolated human fetal liver homegenate sample id: Human fetal liver 5 Sex: Unknown age: 19 postconceptional weeks (pcw) developmental stage: fetal
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Extracted molecule |
total RNA |
Extraction protocol |
Human fetal tissue was dissociated by Collagenase XI enzymatic dissociation for 25 minutes at 37˚C with agitation. Samples were stained with the following primary antibodies, CD235a (349104, FITC, mouse; Biolegend), CD45 (304050, BV711, mouse; Biolegend), EpCAM (324208, APC, mouse; Biolegend), NCAM (362524, PE, mouse; Biolegend) all at 1:100 dilution and incubated for 30mins at 4˚C. DAPI (D1306, ThermoFisher Scientific) at 1:1000 dilution was used for live/dead staining. Cells were sorted using a BD FACS Aria II instrument and deposited as single cells into 96-well plates, pre-loaded with lysis buffer (1% Triton X-100, 1mM dNTP, 1μM oligo-dT30, 1:1.2x106 ERCC ExFold RNA spike-in, Recombinant RNase Inhibitor (2313B, Takara Clontech). Single-cell sequencing was performed using SmartSeq2. RNA was converted into cDNA using SMARTScribe Reverse Transcriptase (639538, Takara Clontech) and amplified for 21 cycles (Kapa HiFi HotStart ReadyMix 2x, KK2602, KAPA Biosystems). Successful single cell libraries were identified by capillary gel electrophoresis (DNF-474-1000, High Sensitivity NGS Fragment Analysis Kit, AATI) and converted into sequencing libraries using a Nextera XT DNA Sample Preparation Kit (FC-131-1096, Illumina). Barcoded libraries were pooled and subjected to 75 base pair paired-end sequencing on a Illumina HiSeq 2500 instrument.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2500 |
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Data processing |
Raw sequencing reads were aligned using STAR and per gene counts were calculated using HTSEQ Gene counts were further analyzed using the R package SCATER for pre-processing, quality control and normalization To filter out bad cells from scRNA-seq analysis we used median absolute deviations (MADs) on library size, total genes detected, mitochondrial gene percentage and artificial ERCC spike in percentage. Two deviations from the median were used to remove cell outliers. For library size and total genes the lower tail outliers were excluded. For mitochondrial and ERCC spike in percentage upper tail outliers were excluded. T-SNE analysis, clustering and gene expression performed in R Genome_build: GRCh38 Supplementary_files_format_and_content: [Series_count_matrix.csv] Raw HTSEQ counts for all samples and feature date [Series_feature_matrix.csv] MetaData including FACS gating population for each sample
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Submission date |
Apr 30, 2019 |
Last update date |
May 21, 2019 |
Contact name |
Joe M Segal |
E-mail(s) |
Joe.segal8@gmail.com
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Organization name |
Kings college London
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Department |
Centre of Stem Cell and Regenerative Medicine
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Lab |
Tamir Rashid
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Street address |
28 floor Tower wing, Great Maze pond
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City |
London |
ZIP/Postal code |
SE1 9RT |
Country |
United Kingdom |
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Platform ID |
GPL16791 |
Series (1) |
GSE130473 |
Single cell analysis of human fetal liver captures the transcriptional profile of hepatobiliary hybrid progenitors |
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Relations |
BioSample |
SAMN11536110 |
SRA |
SRX5770056 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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